Anti-CD28 compositions

ABSTRACT

Provided herein are novel anti-CD28×anti-B7H3 (also referred to as “αCD28×αB7H3”) heterodimeric bispecific antibodies and methods of using such antibodies for the treatment of cancers. Subject αCD28×αB7H3 antibodies are capable of agonistically binding to CD28 costimulatory molecules on T cells and targeting to B7H3 on tumor cells. Thus, such antibodies selectively enhance anti-tumor activity at tumor sites while minimizing peripheral toxicity. The subject antibodies provided herein are particularly useful for enhancing anti-tumor activity when used in combination with other anti-cancer therapies.

PRIORITY

This application is a continuation of U.S. patent application Ser. No.17/407,135, filed Aug. 19, 2021 which claims the benefit of U.S.Provisional Patent Application Nos. 63/067,834, filed Aug. 19, 2020 and63/092,272, filed Oct. 15, 2020 which are hereby incorporated byreference in their entireties.

SEQUENCE LISTING INCORPORATION PARAGRAPH

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 19, 2021, isnamed 067461-5272-WO_SL.txt and is 1,130,417 bytes in size.

BACKGROUND

The natural immune response against tumor dispatches immune effectorcells such as natural killer (NK) cells and T cells to attack anddestroy tumor cells. Tumor infiltrating lymphocytes (TILs) often expressmultiple immune checkpoint receptors (e.g., PD-1, CTLA-4) andcostimulatory receptors (e.g., ICOS, 4-1BB, OX40, GITR, and CD28). TILslose their cytotoxic ability over time due to upregulation of inhibitoryimmune checkpoints. While checkpoint blockade has demonstrated increasedclinical response rates relative to other treatment options, manypatients still fail to achieve a response to checkpoint blockade.Engagement of costimulatory receptors on TILs could provide a positivesignal capable of overcoming negative signals of immune checkpoints.Preclinical and clinical studies of agonistic costimulatory receptorantibodies have indeed demonstrated that agonism of costimulatoryreceptors can result in impressive anti-tumor responses, activating Tcells to attack tumor cells.

It is also important for cancer therapy to enhance anti-tumor activityby specifically destroying tumor cells while minimizing peripheraltoxicity. In this context, it is crucial that only T cells in thepresence of the target tumor cells are provided a costimulatory signal.However, agonism of costimulatory receptors with monospecificfull-length antibodies is likely nondiscriminatory with regards to TILsvs. peripheral T cells vs. autoantigen-reactive T cells that contributeto autoimmune toxicities. For instance, urelumab, a monospecific,nondiscriminatory, pan-4-1BB agonist antibody, exhibited significantliver toxicity in early phase clinical trials (Segal et al., 2016).Thus, there remains a need for novel immune response enhancingcompositions for the treatment of cancers.

SUMMARY

Provided herein are novel anti-CD28 compositions, includinganti-CD28×anti-TAA (e.g., αCD28×αB7H3) antibodies and methods of usingsuch antibodies for the treatment of cancers. Subject anti-CD28×anti-TAAantibodies are capable of agonistically binding to CD28 costimulatorymolecules on T cells and a tumor associated antigen (e.g., B7H3) ontumor cells. Thus, such antibodies selectively enhance anti-tumoractivity at tumor sites while minimizing peripheral toxicity. Thesubject antibodies provided herein are particularly useful incombination with other anti-cancer therapies, including, for example,checkpoint inhibitors. Also provided herein are novel αCD28 and αB7H3binding domains.

In a first aspect, provided herein is a heterodimeric antibodycomprising: a) a first monomer comprising, from N-terminus toC-terminus, a VH1-CH1-linker-VH1-CH1-hinge-CH2-CH3, wherein the VH1s areeach a first variable heavy domain and CH2-CH3 is a first Fc domain; b)a second monomer comprising, from N-terminus to C-terminus, aVH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain andCH2-CH3 is a second Fc domain; and c) a common light chain comprising,from N-terminus to C-terminus, VL-CL, wherein VL is a variable lightdomain and CL is a constant light domain, wherein the common light chainis separately paired with each VH1-CH1 in the first monomer and theVH2-CH1 in the second monomer, wherein the VH1 and the VL together forma first antigen binding domain (ABD), and the VH2 and the VL togetherform a second ABD, wherein one of the first and second ABDs binds humanCD28 and the other of the first and second ABDs bind human B7H3.

In some embodiments, the first ABD binds human CD28 and the second bindshuman B7H3. In certain embodiments, the first ABD binds human B7H3 andthe second binds human CD28.

In some embodiments, the amino acid sequence of the VH1 domain isselected from the group consisting of SEQ ID NO:518, SEQ ID NO:928, SEQID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501,SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ IDNO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515,SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ IDNO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ IDNO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQ ID NO:544,SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ IDNO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558,SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ IDNO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572,SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ IDNO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQID NO:582, SEQ ID NO:583 and SEQ ID NO:584; and wherein the amino acidsequence of the VL domain is SEQ ID NO:874.

In some embodiments, the amino acid sequence of the VH2 domain isselected from the group consisting of SEQ ID NO: 585, SEQ ID NO:870, SEQID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590,SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ IDNO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604,SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ IDNO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618,SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ IDNO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625,SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ IDNO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639,SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ IDNO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQID NO:649, SEQ ID NO:650, and SEQ ID NO:651.

In certain embodiments, the first Fc domain and second Fc domain areeach variant Fc domains.

In some embodiments of the heterodimeric antibody, the first and secondFc domains comprise a set of heterodimerization skew variants selectedfrom the following heterodimerization variants: S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering.In exemplary embodiments, the first and second Fc domains compriseheterodimerization skew variants S364K/E357Q:L368D/K370S.

In some embodiments, the first and second Fc domains each comprise oneor more ablation variants. In some embodiments, the one or more ablationvariants are E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, one of the first or second monomer furthercomprises a pI variant. In some embodiments, the CH1-hinge-CH2-CH3 ofthe second monomer comprises pI variants N208D/Q295E/N384D/Q418E/N421D,wherein numbering is according to EU numbering.

In some embodiments, the CH1-hinge-CH2-CH3 of the second monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the first Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In exemplary embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428L/434S.

In some embodiments, the second monomer comprises the amino acidsequence of SEQ ID NO:1019, the first monomer comprises the amino acidsequence of SEQ ID NO:1020, and the light chain has the amino acidsequence of SEQ ID NO:1021.

In another aspect, provided herein is a heterodimeric antibodycomprising: a) a first monomer comprising, from N-terminus toC-terminus, a VH1-CH1-hinge-CH2-CH3, wherein VH1 is a first variableheavy domain and CH2-CH3 is a first Fc domain; b) a second monomercomprising, from N-terminus to C-terminus, a VH2-CH1-hinge-CH2-CH3,wherein VH2 is a second variable heavy domain and CH2-CH3 is a second Fcdomain; and c) a common light chain comprising, from N-terminus toC-terminus, VL-CL, wherein VL is a variable light domain and CL is aconstant light domain, wherein the first VH domain and the VL domaintogether form a first ABD, and the second VH domain and the VL domaintogether form a second ABD, and wherein one of the first and second ABDsbinds human CD28 and the other of the first and second ABDs bind humanB7H3.

In certain embodiments, the amino acid sequence of the VH1 domain isselected from the group consisting of SEQ ID NO:518, SEQ ID NO:928, SEQID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQ ID NO:501,SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505, SEQ IDNO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ ID NO:510, SEQID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQ ID NO:515,SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520, SEQ IDNO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ ID NO:525, SEQID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQ ID NO:530,SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534, SEQ IDNO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ ID NO:539, SEQID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQ ID NO:544,SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548, SEQ IDNO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ ID NO:553, SEQID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQ ID NO:558,SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562, SEQ IDNO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ ID NO:567, SEQID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQ ID NO:572,SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576, SEQ IDNO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ ID NO:581, SEQID NO:582, SEQ ID NO:583 and SEQ ID NO:584; and wherein the amino acidsequence of the VL domain is SEQ ID NO:874.

In some embodiments, the amino acid sequence of the VH2 domain isselected from the group consisting of SEQ ID NO:585, SEQ ID NO:870, SEQID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590,SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ IDNO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604,SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ IDNO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618,SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ IDNO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625,SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ IDNO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639,SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ IDNO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQID NO:649, SEQ ID NO:650, and SEQ ID NO:651.

In certain embodiments, the first Fc domain and second Fc domain areeach variant Fc domains.

In some embodiments, the first and second Fc domains comprise a set ofheterodimerization skew variants selected from the followingheterodimerization variants: S364K/E357Q:L368D/K370S; S364K:L368D/K370S;S364K:L368E/K370S; D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V,wherein numbering is according to EU numbering. In certain embodiments,the first and second Fc domains comprise heterodimerization skewvariants S364K/E357Q:L368D/K370S.

In some embodiments, the first and second Fc domains each comprise oneor more ablation variants. In certain embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In certain embodiments, one of the first or second monomer furthercomprises a pI variant. In exemplary embodiments, the CH1-hinge-CH2-CH3of the first monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In some embodiments, the CH1-hinge-CH2-CH3 of the first monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the second Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428L/434S.

In another aspect, provided herein is a heterodimeric antibodycomprising: a) a first monomer comprising from N-terminal to C-terminal,VH1-CH1-first domain linker-scFv-second domain linker-CH2-CH3, whereinVH1 is a first variable heavy domain, scFv is an anti-CD28 scFv, andCH2-CH3 is a first Fc domain; b) a second monomer comprising fromN-terminal to C-terminal a VH1-CH1-hinge-CH2-CH3, wherein CH2-CH3 is asecond Fc domain; and c) a light chain comprising, from N-terminus toC-terminus, VL1-CL, wherein VL1 is a variable light domain and CL is aconstant light domain, wherein each of the VH1 domain and the first VL1domain together form a first antigen binding domain (ABD) and the scFvcomprises a second VH domain (VH2), a scFv linker, and a second VLdomain (VL2), and the VH2 and the VL2 form a second ABD, wherein one ofthe first and second ABDs bind human CD28 and the other of the first andsecond ABDs bind a tumor target antigen (TTA).

In certain embodiments, the first ABDs bind human CD28 and the secondABD binds a TTA. In some embodiments, the first ABDs bind a TTA and thesecond ABD binds human CD28.

In some embodiments, the scFv comprises, from N- to C-terminal, VL2-scFvlinker-VH2. In some embodiments, the scFv comprises, from N- toC-terminal, VH2-scFv linker-VL2.

In some embodiments, the amino acid sequence of the VH2 is selected fromthe group consisting of SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591,SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ IDNO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605,SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ IDNO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619,SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ IDNO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626,SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ IDNO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640,SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ IDNO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQID NO:650, and SEQ ID NO:651; and wherein the amino acid sequence of theVL2 is selected from the group consisting of SEQ ID NO:874, SEQ IDNO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661,SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ IDNO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675,SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ IDNO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689,SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ IDNO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703,SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ IDNO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717,SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ IDNO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731,SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ IDNO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745,SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ IDNO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.

In certain embodiments, the TTA is human B7H3.

In some embodiments, the first Fc domain and second Fc domain are eachvariant Fc domains.

In exemplary embodiments, the first and second Fc domains comprise a setof heterodimerization skew variants selected from the followingheterodimerization variants: S364K/E357Q:L368D/K370S; S364K:L368D/K370S;S364K:L368E/K370S; D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V,wherein numbering is according to EU numbering. In some embodiments, thefirst and second Fc domains comprise heterodimerization skew variantsS364K/E357Q:L368D/K370S.

In some embodiments, the first and second Fc domains each comprise oneor more ablation variants. In certain embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In some embodiments, one of the first or second monomer furthercomprises a pI variant. In exemplary embodiments, the CH1-hinge-CH2-CH3of the second monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In exemplary embodiments, the CH1-hinge-CH2-CH3 of the second monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the first Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In certain embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428L/434S.

In one aspect, provided herein is a heterodimeric antibody comprising:a) a first monomer comprising: i) a scFv comprising a first variableheavy domain, an scFv linker and a first variable light domain; and ii)a first Fc domain, wherein the scFv is covalently attached to theN-terminus of the first Fc domain using a domain linker; b) a secondmonomer comprising, from N-terminus to C-terminus, aVH1-CH1-hinge-CH2-CH3, wherein VH is a first variable heavy domain andCH2-CH3 is a second Fc domain; and c) a light chain comprising, fromN-terminus to C-terminus, VL1-CL, wherein VL1 is a variable light domainand CL is a constant light domain, wherein the VH1 and the VL1 togetherform a first ABD and wherein the scFv comprises a second VH domain(VH2), a scFv linker, and a second VL domain (VL2), wherein the VH2 andthe VL2 together form a second ABD, and wherein one of the first ABD andsecond ABD binds CD28 and the other of the first ABD and second ABDbinds a TTA.

In some embodiments, the scFv comprises, from N- to C-terminal, VL2-scFvlinker-VH2. In certain embodiments, the scFv comprises, from N- toC-terminal, VH2-scFv linker-VL2.

In certain embodiments, the second ABD binds to human CD28 wherein theamino acid sequence of the VH2 is selected from the group consisting ofSEQ ID NO: 870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ IDNO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597,SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ IDNO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611,SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ IDNO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198,SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ IDNO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637,SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ IDNO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, and SEQ IDNO:651; and wherein the amino acid sequence of the VL2 is selected fromthe group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658,SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ IDNO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672,SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ IDNO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686,SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ IDNO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700,SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ IDNO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714,SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ IDNO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728,SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ IDNO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742,SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ IDNO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200and SEQ ID NO:756.

In some embodiments, the first Fc domain and second Fc domain are eachvariant Fc domains.

In certain embodiments, the first and second Fc domains comprise a setof heterodimerization skew variants selected from the followingheterodimerization variants: S364K/E357Q:L368D/K370S; S364K:L368D/K370S;S364K:L368E/K370S; D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V,wherein numbering is according to EU numbering. In exemplaryembodiments, the first and second Fc domains comprise heterodimerizationskew variants S364K/E357Q:L368D/K370S.

In certain embodiments, the first and second Fc domains each compriseone or more ablation variants. In some embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In certain embodiments, one of the first or second monomer furthercomprises a pI variant. In exemplary embodiments, the CH1-hinge-CH2-CH3of the second monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In exemplary embodiments, the CH1-hinge-CH2-CH3 of the second monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the first Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428L/434S.

In another aspect, provided herein is a heterodimeric antibodycomprising: a) a first monomer comprising from N-terminal to C-terminal,VH1-CH1-hinge-CH2-CH3-domain linker-scFv, wherein VH1 is a firstvariable heavy domain, scFv is an anti-CD28 scFv, and CH2-CH3 is a firstFc domain; b) a second monomer comprising from N-terminal to C-terminala VH1-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a second Fc domain; and c) alight chain comprising, from N-terminus to C-terminus, VL1-CL, whereinVL1 is a variable light domain and CL is a constant light domain,wherein each of the VH1 domain and the first VL1 domain together form afirst antigen binding domain (ABD) and the scFv comprises a second VHdomain (VH2), a scFv linker, and a second VL domain (VL2), and the VH2and the VL2 together form a second ABD, wherein one of the first andsecond ABDs bind human CD28 and the other of the first and second ABDsbind a tumor target antigen (TTA).

In certain embodiments, the first ABD bind human CD28 and the second ABDbinds a TTA. In some embodiments, the first ABD bind a TTA and thesecond ABD binds human CD28.

In certain embodiments, the scFv comprises, from N- to C-terminal,VL2-scFv linker-VH2. In some embodiments, the scFv comprises, from N- toC-terminal, VH2-scFv linker-VL2.

In certain embodiments, the amino acid sequence of the VH2 is selectedfrom the group consisting of SEQ ID NO:870, SEQ ID NO:585, SEQ IDNO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595,SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ IDNO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609,SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ IDNO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623,SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ IDNO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635,SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ IDNO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649,SEQ ID NO:650, and SEQ ID NO:651; and wherein the amino acid sequence ofthe VL2 is selected from the group consisting of SEQ ID NO:874, SEQ IDNO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661,SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ IDNO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675,SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ IDNO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689,SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ IDNO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703,SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ IDNO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717,SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ IDNO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731,SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ IDNO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745,SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ IDNO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.

In some embodiments, the TTA is human B7H3.

In some embodiments, the first Fc domain and second Fc domain are eachvariant Fc domains. In some embodiments, the first and second Fc domainscomprise a set of heterodimerization skew variants selected from thefollowing heterodimerization variants: S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering.In certain embodiments, the first and second Fc domains compriseheterodimerization skew variants S364K/E357Q:L368D/K370S.

In some embodiments, the first and second Fc domains each comprise oneor more ablation variants. In certain embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In some embodiments, one of the first or second monomer furthercomprises a pI variant. In exemplary embodiments, the CH1-hinge-CH2-CH3of the second monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In some embodiments, the CH1-hinge-CH2-CH3 of the second monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the first Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In exemplary embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428L/434S.

In another aspect, provided herein is a nucleic acid compositioncomprising: a) a first nucleic acid encoding the first monomer of any ofthe heterodimeric antibodies described herein; b) a second nucleic acidencoding the second monomer of the heterodimeric antibody; and c) athird nucleic acid encoding the light chain of the heterodimericantibody, respectively. Also provided herein are expression vectorcompositions that include expression vectors comprising one or more ofthe first, second and third nucleic acids, host cells that include suchexpression vector compositions, and methods of making the heterodimericantibodies described herein.

In another aspect, provided herein is a method of treating cancer in apatient in need thereof, comprising administering to the patient aheterodimeric antibody provided herein.

In another aspect, provided herein is a method of treating cancer in apatient in need thereof, comprising administering to the patient: a) aheterodimeric antibody described herein, wherein the TTA is human B7H3;and b) a bispecific antibody that binds CD3 and B7H3.

In yet another aspect, provided herein is a method of treating cancer ina patient in need thereof, comprising administering to the patient: a) aheterodimeric antibody described herein, wherein the TTA is human B7H3;and b) a checkpoint inhibitor selected from the group consisting of ananti-PD-1 antibody and an anti-PD-L1 antibody.

In yet another aspect, provided herein is a composition comprising ananti-CD28 ABD comprising: a) a variable heavy domain with an amino acidsequence selected from the group consisting of SEQ ID NO: 870, SEQ IDNO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589, SEQID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ ID NO:594,SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQ IDNO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603, SEQID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ ID NO:608,SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQ IDNO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617, SEQID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ ID NO:622,SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199, SEQ IDNO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634,SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ IDNO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648,SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652, SEQ IDNO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 and SEQ IDNO:672; and b) variable light domain with an amino acid sequenceselected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657,SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ IDNO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671,SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ IDNO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685,SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ IDNO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699,SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ IDNO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713,SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ IDNO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727,SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ IDNO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741,SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ IDNO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755,SEQ ID NO:1200 and SEQ ID NO:756. In some embodiments, the compositionis an antibody comprising: a) a heavy chain comprising theVH—CH1-hinge-CH2-CH3; and b) a light chain comprising the VL-CL.

In another aspect, provided herein is a composition comprising ananti-B7H3 ABD comprising: a) a variable heavy domain with an amino acidsequence selected from the group consisting of SEQ ID NO:518, SEQ IDNO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500, SEQID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQ ID NO:504, SEQ ID NO:505,SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508, SEQ ID NO:509, SEQ IDNO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ ID NO:513, SEQ ID NO:514, SEQID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQ ID NO:519, SEQ ID NO:520,SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523, SEQ ID NO:524, SEQ IDNO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ ID NO:528, SEQ ID NO:529, SEQID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQ ID NO:533, SEQ ID NO:534,SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537, SEQ ID NO:538, SEQ IDNO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ ID NO:542, SEQ ID NO:543, SEQID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:548,SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551, SEQ ID NO:552, SEQ IDNO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ ID NO:556, SEQ ID NO:557, SEQID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQ ID NO:561, SEQ ID NO:562,SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565, SEQ ID NO:566, SEQ IDNO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ ID NO:570, SEQ ID NO:571, SEQID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQ ID NO:575, SEQ ID NO:576,SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579, SEQ ID NO:580, SEQ IDNO:581, SEQ ID NO:582, SEQ ID NO:583 and SEQ ID NO:584; and b) variablelight domain having the amino acid sequence selected from the groupconsisting of SEQ ID NO:874 and SEQ ID NO: 932.

In one aspect, provided herein is composition comprising an anti-B7H3ABD comprising: a) a variable heavy domain having the amino acidsequence of SEQ ID NO:946; and b) a variable light domain having theamino acid sequence of SEQ ID NO:950.

In another aspect, provided herein is composition comprising ananti-B7H3 ABD comprising: a) a variable heavy domain having the aminoacid sequence of SEQ ID NO:956; and b) a variable light domain havingthe amino acid sequence of SEQ ID NO:960.

In one aspect, provided herein is a composition comprising an anti-B7H3ABD comprising: a) a variable heavy domain having the amino acidsequence of SEQ ID NO:964; and b) a variable light domain having theamino acid sequence of SEQ ID NO:968.

In another aspect, provided herein is a composition comprising ananti-B7H3 ABD comprising: a) a variable heavy domain having the aminoacid sequence of SEQ ID NO:972; and b) a variable light domain havingthe amino acid sequence of SEQ ID NO:976.

In some embodiments, the composition is an antibody comprising: a) aheavy chain comprising the VH linked to —CH1-hinge-CH2-CH3; and b) alight chain comprising the VL linked to —CL.

In another aspect, provided herein is a nucleic acid compositioncomprising: a) a first nucleic acid encoding the VH of any of theanti-CD28 ABDs or anti-B7H3 ABDs described herein; and b) a secondnucleic acid encoding the VL of the anti-CD28 or anti-B7H3 ABD,respectively. Also provided herein are expression vector compositionsthat include expression vectors comprising one or more of the first, andsecond nucleic acids, host cells that include such nucleic acidcompositions or expression vector compositions, and methods of makingthe anti-CD28 ABDs or anti-B7H3 ABDs compositions described herein.

In one aspect, provided herein is a composition that includes a CD28antigen binding domain (ABD). The CD28 ABD includes the variable heavycomplementary determining regions 1-3 (vhCDR1-3) and the variable lightcomplementary determining regions (vlCDR1-3) of any of the followingCD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1,1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71,CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1,341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1.

In some embodiments, the CD28 ABD includes a variable heavy domain and avariable light domain of any of the following CD28 binding domains:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1. In exemplaryembodiments, the CD28 antigen binding domain selected from the followingCD28 antigen binding domain: CD28 binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,hu9.3[CD28]_H1L1.

In another aspect, provided herein is an anti-CD28 antibody thatincludes a CD28 antigen binding domain (ABD). The CD28 antigen bindingdomain includes the variable heavy complementary determining regions 1-3(vhCDR1-3) and the variable light complementary determining regions(vlCDR1-3) of any of the following CD28 binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,hu9.3[CD28]_H1L1. In some embodiments, the CD28 ABD includes a variableheavy domain and a variable light domain of any of the following CD28binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1. In exemplaryembodiments, the CD28 antigen binding domain selected from the followingCD28 antigen binding domain: CD28 binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,hu9.3[CD28]_H1L1.

In some embodiments, the anti-CD28 antibody includes: a) a first monomerthat includes a first antigen binding domain and a first constantdomain; and b) a second monomer that includes a second antigen bindingdomain and a second constant domain, wherein either of the first antigenbinding domain or second antigen binding domain is the CD28 antigenbinding domain.

In some embodiments, the first antigen binding domain and the secondantigen binding domain bind different antigens.

In certain embodiments, the CD28 antigen binding domain is an anti-CD28single chain fragment (scFv). In exemplary embodiments, the scFvincludes a charged scFv linker.

In some embodiments, the first and second constant domains each includeCH2-CH3. In exemplary embodiments, the first and second constant domainseach are a variant constant domain. In certain embodiments, the firstand second constant domains include a set of heterodimerization variantsselected from the group consisting of S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V. In certain embodiments, the first and secondmonomers each further include one or more ablation variants. Inexemplary embodiments, the ablation variants areE233P/L234V/L235A/G236del/S267K. In some embodiments, at least one ofthe first or second monomer further include one or more pI variants. Inparticular embodiments, the pI variants areN208D/Q295E/N384D/Q418E/N421D.

In another aspect, provided herein is a composition that includes a B7H3antigen binding domain (ABD). The B7H3 binding domain includes thevariable heavy complementary determining regions 1-3 (vhCDR1-3) and thevariable light complementary determining regions (vlCDR1-3) of any ofthe following B7H3 binding domains: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704.

In some embodiments, the B7H3 ABD includes a variable heavy domain and avariable light domain of any of the following B7H3 binding domains:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In exemplary embodiments, the B7H3 ABD is selected from the followingB7H3 antigen binding domain: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704.

In yet another aspect, provided herein is an anti-B7H3 antibody thatincludes an B7H3 antigen binding domain, the B7H3 antigen binding domainincludes the variable heavy complementary determining regions 1-3(vhCDR1-3) and the variable light complementary determining regions(vlCDR1-3) of any of the following B7H3 antigen binding domain:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In some embodiments, the anti-B7H3 antibody includes a B7H3 antigenbinding domain. The B7H3 antigen binding domain includes a variableheavy domain and a variable light domain of any of the following B7H3antigen binding domains: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704. In exemplary embodiments,the B7H3 antigen binding domain selected from any one of the followingB7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704.

In some embodiments, the antibody includes: a) a first monomer thatincludes a first antigen binding domain and a first constant domain; andb) a second monomer that includes a second antigen binding domain and asecond constant domain, wherein either of the first antigen bindingdomain or second antigen binding domain is the B7H3 antigen bindingdomain. In certain embodiments, first antigen binding domain and thesecond antigen binding domain bind different antigens.

In exemplary embodiments, the first antigen binding domain is a B7H3antigen binding domain and the second antigen binding domain is a CD28binding domain. In some embodiments, the CD28 binding domain includesthe vhCDR1-3, and vlCDR1-3 of any of the following CD28 binding domains:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1. In someembodiments, the CD28 binding domain includes the variable heavy domainand variable light domain of any of the following CD28 binding domains:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1. In certainembodiments, the CD28 binding domain is an anti-CD28 scFv. In exemplaryembodiments, the scFv comprises a charged scFv linker.

In some embodiments, the first and second constant domains each compriseCH2-CH3. In exemplary embodiments, the first and second constant domainseach are a variant constant domain.

In particular embodiments, the first and second constant domains includea set of heterodimerization variants selected fromS364K/E357Q:L368D/K370S; S364K:L368D/K370S; S364K:L368E/K370S;D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V. In certainembodiments, the first and second monomers each include one or moreablation variants. In certain embodiments, the ablation variants areE233P/L234V/L235A/G236del/S267K. In some embodiments, at least one ofthe first or second monomers further include one or more pI variants. Inparticular embodiments, the pI variants areN208D/Q295E/N384D/Q418E/N421D.

In another aspect, provided herein is an anti-CD28×anti-TAA 1+1Fab-scFv-Fc heterodimeric antibody. In one embodiment, the heterodimericantibody includes: a) a first monomer comprising: i) an anti-CD28 scFvcomprising a first variable heavy domain, an scFv linker and a firstvariable light domain; and ii) a first Fc domain, wherein the scFv iscovalently attached to the N-terminus of the first Fc domain using adomain linker; b) a second monomer comprising, from N-terminus toC-terminus, a VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variableheavy domain and CH2-CH3 is a second Fc domain; and c) a third monomercomprising a second variable light domain, wherein the second variableheavy domain and the second variable light domain form a tumorassociated antigen (TAA) binding domain.

In some embodiments, the anti-CD28 scFv comprises the vhCDR1-3 and thevlCDR1-3 of any of the following CD28 antigen binding domains:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.

In certain embodiments, the first variable heavy domain and firstvariable light domain of the anti-CD28 scFv are the variable heavydomain and variable light domain, respectively, of any of the followingCD28 antigen binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1,1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71,CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1,341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, andhu9.3[CD28]_H1L1.

In particular embodiments, the TAA binding domain is a B7H3 bindingdomain. In some embodiments, the B7H3 binding domain comprises thevhCDR1-3 and vlCDR1-3 of any of the following B7H3 antigen bindingdomains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In exemplary embodiments, the second variable heavy domain and thesecond variable light domain are the variable heavy domain and variablelight domain, respectively, of any of the following B7H3 antigen bindingdomains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In exemplary embodiments, the anti-CD28 scFv is oriented, fromN-terminus to C-terminus, first variable light domain-scFv linker-firstvariable heavy domain. In other embodiments, the anti-CD28 scFv isoriented, from N-terminus to C-terminus, first variable heavydomain-scFv linker-first variable light domain. In many embodiments, thescFv linker is a charged scFv linker.

In certain embodiments, first and second Fc domains are variant Fcdomains. In some embodiments, the first and second Fc domains comprise aset of heterodimerization skew variants selected from the groupconsisting of S364K/E357Q:L368D/K370S; S364K:L368D/K370S;S364K:L368E/K370S; D401K:T411E/K360E/Q362E; and T366W:T366S/L368A/Y407V,wherein numbering is according to EU numbering. In exemplaryembodiments, the first and second Fc domains comprise heterodimerizationskew variants S364K/E357Q:L368D/K370S.

In certain embodiments, first and second Fc domains each comprise one ormore ablation variants. In exemplary embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In some embodiments, one of the first or second monomers comprise one ormore pI variants. In exemplary embodiments, the CH1-hinge-CH2-CH3 of thesecond monomer comprises pI variants N208D/Q295E/N384D/Q418E/N421D,wherein numbering is according to EU numbering.

In exemplary embodiments, the first Fc domain comprises amino acidvariants S364K/E357Q/E233P/L234V/L235A/G236del/S267K; theCH1-hinge-CH2-CH3 of the second monomer comprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and wherein numbering is according to EU numbering.

In certain embodiments, the scFv linker is a charged scFv linker havingthe amino acid sequence (GKPGS)₄.

In particular embodiments, the first and second Fc domains each furthercomprise amino acid variants 428/434S.

In some embodiments, the anti-CD28×anti-TAA 1+1 Fab-scFv-Fcheterodimeric antibody includes: a) a first monomer comprising, fromN-terminus to C-terminus, an anti-CD28 scFv-linker-CH2-CH3, whereinCH2-CH3 is a first Fc domain; b) a second monomer comprising, fromN-terminus to C-terminus, a VH—CH1-hinge-CH2-CH3, wherein CH2-CH3 is asecond variant Fc domain; and c) a third monomer comprising VL-CL;wherein the first variant Fc domain comprises amino acid variantsS364K/E357Q, wherein the second variant Fc domain comprises amino acidvariants L368D/K370S, wherein the first and second variant Fc domainseach comprises amino acid variants E233P/L234V/L235A/G236del/S267K,wherein the CH1-hinge-CH2-CH3 of the second monomer comprises amino acidvariants N208D/Q295E/N384D/Q418E/N421D, wherein the VH and VL form atumor associated antigen (TAA) binding domain, and wherein the anti-CD28scFv comprises the variable heavy domain and the variable light domainof one of the following CD28 antigen binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,and hu9.3[CD28]_H1L1, and wherein numbering is according to EUnumbering.

In certain embodiments, the TAA binding domain is a B7H3 binding domain.In some embodiments, VH and VL are the variable heavy domain andvariable light domain, respectively, of any of the following B7H3antigen binding domains: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704.

In exemplary embodiments, the scFv comprises a charged scFv linkerhaving the amino acid sequence (GKPGS)₄. In some embodiments, the firstand second variant Fc domains each further comprise amino acid variants428/434S, wherein numbering is according to EU numbering.

In another aspect, provided herein are anti-CD28×anti-TAA 2+1Fab₂-scFv-Fc antibodies that include: a) a first monomer comprising,from N-terminus to C-terminus, a VH1-CH1-linker 1-anti-CD28 scFv-linker2-CH2-CH3, wherein VH1 is a first variable heavy domain, linker 1 andlinker 2 are a first domain linker and second domain linker,respectively, and CH2-CH3 is a first Fc domain; b) a second monomercomprising, from N-terminus to C-terminus, a VH2-CH1-hinge-CH2-CH3,wherein VH2 is a second variable heavy domain and CH2-CH3 is a second Fcdomain; and c) a common light chain comprising a variable light domain;wherein the first variable heavy domain and the variable light domainform a first tumor associated antigen (TAA) binding domain, and thesecond variable heavy domain and the variable light domain form a secondTAA binding domain.

In exemplary embodiments, the first TAA binding domain and second TAAbinding domain are each B7H3 binding domains. In exemplary embodiments,the first and second B7H3 binding domains each comprise the vhCDR1-3 andvlCDR1-3 of any of the following B7H3 antigen binding domains:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704. In some embodiments, the first and second variable heavy domaineach comprise a variable heavy domain of a B7H3 binding domain, and thevariable light domain comprises a variable light domain of the B7H3binding domain, wherein the B7H3 binding domain is any of the followingB7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704.

In several embodiments of the anti-CD28×anti-TAA 2+1 Fab₂-scFv-Fcantibody, the anti-CD28 scFv comprises an scFv variable heavy domain, anscFv variable light domain and an scFv linker that connects the scFvvariable heavy domain and the scFv variable light domain. In certainembodiments, the anti-CD28 scFv comprises the vhCDR1-3 and the vlCDR1-3of any of the following CD28 antigen binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,and hu9.3[CD28]_H1L1. In certain embodiments, the scFv variable heavydomain and the scFv variable light domain of the anti-CD28 scFvcomprises the variable heavy domain and variable light domain,respectively, of any of the following CD28 antigen binding domains:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.

In some embodiments, the scFv variable heavy domain is attached to theC-terminus of the CH1 of the first monomer using the first domain linkerand the scFv variable light domain is covalently attached to theN-terminus of the first Fc domain using the second domain linker. Inother embodiments, the scFv variable light domain is attached to theC-terminus of the CH1 of the first monomer using the first domain linkerand the scFv variable heavy domain is covalently attached to theN-terminus of the first Fc domain using the second domain linker. Insome embodiments, the scFv linker is a charged scFv linker.

In certain embodiments, the first and second Fc domains are variantconstant domains. In the first and second Fc domains comprise a set ofheterodimerization variants selected from the followingheterodimerization skew variants: S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering.In some embodiments, the first and second Fc domains includeheterodimerization skew variants S364K/E357Q:L368D/K370S.

In some embodiments, the first and second Fc domains each include one ormore ablation variants. In exemplary embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In some embodiments, one of the first or second monomer comprises one ormore pI variants. In particular embodiments, the CH1-hinge-CH2-CH3 ofthe second monomer comprises pI variants N208D/Q295E/N384D/Q418E/N421D,wherein numbering is according to EU numbering.

In exemplary embodiments, the first Fc domain of the first monomercomprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, the CH1-hinge-CH2-CH3 ofthe second monomer comprises amino acid variantsN208D/E233P/L234V/L235A/G236del/S267K/Q295E/L368D/K370S/N384D/Q418E/N421D,and wherein numbering is according to EU numbering.

In some embodiments, the anti-CD28 scFv comprises a charged scFv linkerhaving the amino acid sequence (GKPGS)₄. In certain embodiments, thefirst and second variant Fc domains each further comprise amino acidvariants 428/434S, wherein numbering is according to EU numbering.

In some embodiments, the anti-CD28×anti-TAA 2+1 Fab₂-scFv-Fc antibodiesinclude: a) a first monomer comprising from N-terminal to C-terminal, aVH1-CH1-linker 1-anti-CD28 scFv-linker 2-CH2-CH3, wherein CH2-CH3 is afirst variant Fc domain; b) a second monomer comprising from N-terminalto C-terminal a VH1-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a secondvariant Fc domain; and c) a common light chain comprising VL-CL; whereinthe first variant Fc domain comprises amino acid variants S364K/E357Q,wherein the second variant Fc domain comprises amino acid variantsL368D/K370S, wherein the first and second variant Fc domains eachcomprises amino acid variants E233P/L234V/L235A/G236del/S267K, whereinthe CH1-hinge-CH2-CH3 of the second monomer comprises amino acidvariants N208D/Q295E/N384D/Q418E/N421D, wherein the VH1 and VL each forma tumor associated antigen (TAA) binding domain, wherein the anti-CD28scFv comprises the variable heavy domain and the variable light domainof any of the following CD28 antigen binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,and hu9.3[CD28]_H1L1, and wherein numbering is according to EUnumbering.

In some embodiments, the VH1 and VL form a B7H3 binding domain. Inexemplary embodiments, the VH1 and VL are the variable heavy domain andvariable light domain of any of the following B7H3 antigen bindingdomains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In some embodiments, the scFv comprises a charged scFv linker having theamino acid sequence (GKPGS)₄. In certain embodiments, the first andsecond variant Fc domains each further comprise amino acid variants428/434S.

In another aspect, provided herein are anti-CD28×anti-TAA 1+1 CLCheterodimeric antibodies that include: a) a first monomer comprising,from N-terminus to C-terminus, a VH1-CH1-hinge-CH2-CH3, wherein VH1 is afirst variable heavy domain and CH2-CH3 is a first Fc domain; b) asecond monomer comprising, from N-terminus to C-terminus, aVH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variable heavy domain andCH2-C3 is a second Fc domain; and c) a common light chain comprising,from N-terminus to C-terminus, VL-CL, wherein VL is a variable lightdomain and CL is a constant light domain, wherein the first variableheavy domain and the variable light domain form a first antigen bindingdomain, and the second variable heavy domain and the variable lightdomain form a second antigen binding domain.

In some embodiments, the first Fc domain and second Fc domain are eachvariant Fc domains. In certain embodiments, the first and second Fcdomains comprise a set of heterodimerization skew variants selected fromthe following heterodimerization variants: S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering.In exemplary embodiments, the first and second Fc domains compriseheterodimerization skew variants S364K/E357Q:L368D/K370S.

In certain embodiments, the first and second Fc domains each compriseone or more ablation variants. In exemplary embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In some embodiments, one of the first or second monomer furthercomprises a pI variant. In particular embodiments, the CH1-hinge-CH2-CH3of the first monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In certain embodiments, the CH1-hinge-CH2-CH3 of the first monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,the second Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, and wherein numbering isaccording to EU numbering.

In some embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428/434S.

In certain embodiments, the first antigen binding domain or the secondbinding domain binds CD28 and the other antigen binding domain binds atumor associated antigen (TAA).

In certain embodiments, the second antigen binding domain binds CD28 andVH2 and VL comprises the variable heavy domain and variable lightdomain, respectively, of any one of the following CD28 binding domains:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1.

In some embodiments, the first antigen binding domain binds the TTA. INexemplary embodiments, the TAA is B7H3. In exemplary embodiments, theVH1 and VL comprises the variable heavy domain and variable lightdomain, respectively, of any one of the following B7H3 binding domains:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In exemplary embodiments, the first antigen binding domain binds B7H3and the second antigen binding domain binds CD28, VH1 is variable heavydomain 2E4A3.189[B7H3]_H1.22, VH2 is variable heavy domainA7[CD28]_H1.14, and VL is variable light domain 1A7[CD28]_L1.

In one aspect, provided herein are anti-CD28×anti-TAA 2+1 CLCheterodimeric antibodies that include: a) a first monomer comprising,from N-terminus to C-terminus, a VH1-CH1-linker-VH1-CH1-hinge-CH2-CH3,wherein the VH1s are each a first variable heavy domain and CH2-CH3 is afirst Fc domain; b) a second monomer comprising, from N-terminus toC-terminus, a VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variableheavy domain and CH2-C3 is a second Fc domain; and c) a common lightchain comprising, from N-terminus to C-terminus, VL-CL, wherein VL is avariable light domain and CL is a constant light domain, wherein thefirst variable heavy domains and the variable light domain each form afirst antigen binding domain, and the second variable heavy domain andthe variable light domain form a second antigen binding domain.

In some embodiments, the first Fc domain and second Fc domain are eachvariant Fc domains. In some embodiments, the first and second Fc domainscomprise a set of heterodimerization skew variants selected from thefollowing heterodimerization variants: S364K/E357Q:L368D/K370S;S364K:L368D/K370S; S364K:L368E/K370S; D401K:T411E/K360E/Q362E; andT366W:T366S/L368A/Y407V, wherein numbering is according to EU numbering.In certain embodiments, the first and second Fc domains compriseheterodimerization skew variants S364K/E357Q:L368D/K370S.

In several embodiments, the first and second Fc domains each compriseone or more ablation variants. In exemplary embodiments, the one or moreablation variants are E233P/L234V/L235A/G236del/S267K, wherein numberingis according to EU numbering.

In particular embodiments, the one of the first or second monomerfurther comprises a pI variant. In exemplary embodiments, theCH1-hinge-CH2-CH3 of the first monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In some embodiments of the anti-CD28×anti-TAA 2+1 CLC heterodimericantibodies, the CH1-hinge-CH2-CH3 of the first monomer comprises aminoacid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,the second Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, and wherein numbering isaccording to EU numbering.

In some embodiments, the first and second variant Fc domains eachcomprise amino acid variants 428/434S.

In certain embodiments, the first antigen binding domains binds CD28 andthe second antigen binding domain binds a tumor associated antigen(TAA). In exemplary embodiments, VH1 and VL comprises the variable heavydomain and variable light domain, respectively, of any one of thefollowing CD28 binding domains: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1,1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71,CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1,341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, andhu9.3[CD28]_H1L1.

In some embodiments, the TAA is B7H3. In exemplary embodiments, VH2 andVL comprises the variable heavy domain and variable light domain,respectively, of any one of the following B7H3 binding domains:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704.

In some embodiments, VH1 is variable heavy domain 1A7[CD28]_H1.14, VH2is variable heavy domain 2E4A3.189[B7H3]_H1.22, and VL is variable lightdomain 1A7[CD28]_L1.

In another aspect, provided herein is a heterodimeric antibody selectedfrom the following heterodimeric antibodies: XENP34730, XENP34389,XENP34728, XENP34717 and XENP34339.

Also provided herein are nucleic acid compositions encoding thecompositions and antibodies provided herein, expression vectors thatinclude such nucleic acids, and host cells that include the expressionvectors.

In another aspect, provided herein are methods of treating a cancercomprising administering to a patient in need thereof an antibodyprovided herein (e.g., an anti-CD28×anti-TAA antibody). In someembodiments, the patient is also administered a cancer therapeutic. Inparticular embodiments, the therapeutic is a checkpoint inhibitor (e.g.,an anti-PD1 antibody) or an anti-CD3×anti-TAA bispecific antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sequences for human, mouse, and cynomolgus CD28. SuchCD28 are useful for the development of cross-reactive CD28 antigenbinding domains for ease of clinical development.

FIGS. 2A and 2B depict the sequences for human, mouse, and cynomolgusB7H3. Such B7H3 are useful for the development of cross-reactive B7H3antigen binding domains for ease of clinical development.

FIG. 3A-3F depict useful pairs of heterodimerization variant sets(including skew and pI variants). In FIG. 3F, there are variants forwhich there are no corresponding “monomer 2” variants. Such variants arepI variants that can be used alone on either monomer of a αB7H3ΔαCD28bsAb, or included, for example, on the non-scFv side of a format thatutilizes an scFv as a component and an appropriate charged scFv linkercan be used on the second monomer that utilizes an scFv as the CD28binding domain. Suitable charged linkers are shown in FIG. 6 .

FIG. 4 depicts a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These variants can beoptionally and independently combined with other variants, includingheterodimerization variants, outlined herein.

FIG. 5 depict useful ablation variants that ablate FcγR binding (alsoreferred to as “knockouts” or “KO” variants). In some embodiments, suchablation variants are included in the Fc domain of both monomers of thesubject antibody described herein. In other embodiments, the ablationvariants are only included on only one variant Fc domain.

FIG. 6 depicts a number of charged scFv linkers that find use inincreasing or decreasing the pI of the subject heterodimeric αB7H3ΔαCD28bsAbs that utilize one or more scFv as a component, as described herein.The (+H) positive linker finds particular use herein, particularly withanti-CD28 V_(L) and V_(H) sequences shown herein. A single prior artscFv linker with a single charge is referenced as “Whitlow”, fromWhitlow et al., Protein Engineering 6(8):989-995 (1993). It should benoted that this linker was used for reducing aggregation and enhancingproteolytic stability in scFvs. Such charged scFv linkers can be used inany of the subject antibody formats disclosed herein that include scFvs(e.g., 1+1 Fab-scFv-Fc and 2+1 Fab₂-scFv-Fc formats).

FIG. 7 depicts a number of exemplary domain linkers. In someembodiments, these linkers find use linking a single-chain Fv to an Fcchain. In some embodiments, these linkers may be combined in anyorientation. For example, a GGGGS linker may be combined with a “lowerhalf hinge” linker at the N-terminus or at the C-terminus. In someembodiments, two or more of the domain linkers depicted in FIG. 7 can becombined to form longer domain linkers for use in the heterodimericantibodies described herein.

FIG. 8 shows a particularly useful embodiment of the heterodimeric Fcdomains (i.e. CH2-CH3 in this embodiment) of the αB7H3ΔαCD28 bsAbs ofthe invention.

FIG. 9 depicts various heterodimeric skewing variant amino acidsubstitutions that can be used with the heterodimeric antibodiesdescribed herein.

FIGS. 10A-10C show the sequences of several useful heterodimericαB7-H3×αCD28 bsAb backbones based on human IgG1, without the cytokinesequences. Heterodimeric Fc backbone 1 is based on human IgG1 (356E/358Mallotype), and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants on both chains.Heterodimeric Fc backbone 2 is based on human IgG1 (356E/358M allotype),and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K skew variant on a second heterodimeric Fc chain, and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Heterodimeric Fc backbone 3 is based on human IgG1 (356E/358M allotype),and includes the L368E/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K skew variant on a second heterodimeric Fc chain, and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Heterodimeric Fc backbone 4 is based on human IgG1 (356E/358M allotype),and includes the K360E/Q362E/T411E skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the D401K skew variant on a second heterodimeric Fc chain, and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Heterodimeric Fc backbone 5 is based on human IgG1 (356D/358L allotype),and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants on both chains.Heterodimeric Fc backbone 6 is based on human IgG1 (356E/358M allotype),and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants and N297A variantthat removes glycosylation on both chains. Heterodimeric Fc backbone 7is based on human IgG1 (356E/358M allotype), and includes theL368D/K370S skew variants and the Q295E/N384D/Q418E/N421D pI variants ona first heterodimeric Fc chain, the S364K/E357Q skew variants on asecond heterodimeric Fc chain, and the E233P/L234V/L235A/G236del/S267Kablation variants and N297S variant that removes glycosylation on bothchains. Heterodimeric Fc backbone 8 is based on human IgG4, and includesthe L368D/K370S skew variants and the Q295E/N384D/Q418E/N421D pIvariants on a first heterodimeric Fc chain, the S364K/E357Q skewvariants on a second heterodimeric Fc chain, and the S228P (according toEU numbering, S241P in Kabat) variant that ablates Fab arm exchange (asis known in the art) on both chains. Heterodimeric Fc backbone 9 isbased on human IgG2, and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain.Heterodimeric Fc backbone 10 is based on human IgG2, and includes theL368D/K370S skew variants and the Q295E/N384D/Q418E/N421D pI variants ona first heterodimeric Fc chain, the S364K/E357Q skew variants on asecond heterodimeric Fc chain, and the S267K ablation variant on bothchains. Heterodimeric Fc backbone 11 is based on human IgG1 (356E/358Mallotype), and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants and M428L/N434SXtend variants on both chains. Heterodimeric Fc backbone 12 is based onhuman IgG1 (356E/358M allotype), and includes the L368D/K370S skewvariants on a first heterodimeric Fc chain, the S364K/E357Q skewvariants and P217R/P229R/N276K pI variants on a second heterodimeric Fcchain, and the E233P/L234V/L235A/G236del/S267K ablation variants on bothchains.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition or as an alternative to the skew, pI andablation variants contained within the backbones of this Figure.Additionally, the backbones depicted herein may include deletion of theC-terminal glycine (K446_) and/or lysine (K447_). The C-terminal glycineand/or lysine deletion may be intentionally engineered to reduceheterogeneity or in the context of certain bispecific formats, such asthe mAb-scFv format. Additionally, C-terminal glycine and/or lysinedeletion may occur naturally for example during production and storage.

FIG. 11 depicts illustrative sequences of heterodimeric B7H3×CD28 bsAbbackbone for use in the 2+1 mAb-scFv format. The format depicted here isbased on heterodimeric Fc backbone 1 as depicted in Figure X, exceptfurther including G446_on monomer 1 (−) and G446_/K447_on monomer 2 (+).It should be noted that any of the additional backbones depicted inFigure X may be adapted for use in the 2+1 mAb-scFv format with orwithout including K447_on one or both chains. It should be noted thatthese sequences may further include the M428L/N434S variants.

FIG. 12 depicts sequences for “CH1+ hinge” that find use in embodimentsof αB7H3ΔαCD28 bsAbs that utilize a Fab a binding domain. The “CH1+hinge” sequences find use linking the variable heavy domain (V_(H)) tothe Fc backbones (as depicted in FIG. 39 ). For particular embodimentswherein the Fab is on the (+) side, the “CH1(+)+ hinge” sequences mayfind use. For particular embodiments wherein the Fab is on the (−) side,the “CH1(−)+ hinge” sequences may find use.

FIG. 13 depicts sequences for “CH1+ half hinge” domain linker that finduse in embodiments of αB7H3ΔαCD28 bsAbs in the 2+1 Fab₂-scFv-Fc formator 2+1 CLC format. In the 2+1 Fab₂-scFv-Fc format, the “CH1+ half hinge”sequences find use linking the variable heavy domain (V_(H)) to the scFvdomain on the Fab-scFv-Fc side of the bispecific antibody. In the 2+1CLC format, the “CH1+ half hinge” sequences find use linking the firstvariable heavy domain (V_(H)) to the second V_(H) domain on theFab-Fab-Fc side of the bispecific antibody. It should be noted thatother linkers may be used in place of the “CH1+ half hinge”. It shouldalso be noted that although the sequences here are based on the IgG1sequence, equivalents can be constructed based on the IgG2 or IgG4sequences.

FIG. 14 depicts sequences for “CH1” that find use in embodiments ofαB7H3ΔαCD28 bsAbs.

FIG. 15 depicts sequences for “hinge” that find use in embodiments ofαB7H3ΔαCD28 bsAbs.

FIG. 16 depicts the constant domain of the cognate light chains whichfind use in the subject αB7H3ΔαCD28 bsAbs that utilize a Fab bindingdomain.

FIG. 17 depicts the sequences for XENP16432, an anti-PD-1 mAb based onnivolumab and IgG1 backbone with E233P/L234V/L235A/G236del/S267Kablation variant. CDRs are underlined and slashes indicate the border(s)between the variable regions and constant domain.

FIG. 18 depicts the variable heavy and variable light chain sequencesfor 1A7, an exemplary phage-derived CD28 binding domain, as well as thesequences for XENP28428, an anti-CD28 mAb based on 1A7 and IgG1 backbonewith E233P/L234V/L235A/G236del/S267K ablation variant. CDRs areunderlined and slashes indicate the border(s) between the variableregions and constant domain. As noted herein and is true for everysequence herein containing CDRs, the exact identification of the CDRlocations may be slightly different depending on the numbering used asis shown in Table 2, and thus included herein are not only the CDRs thatare underlined but also CDRs included within the V_(H) and V_(L) domainsusing other numbering systems. Furthermore, as for all the sequences inthe Figures, these V_(H) and V_(L) sequences can be used either in ascFv format or in a Fab format.

FIG. 19 depicts the sequence for illustrative affinity-optimized 1A7 VHvariants. It should be noted that the VH depicted herein can be pairedwith any of the other variable light domains depicted herein.

FIG. 20 depicts the sequence for illustrative affinity-optimized1A7-derived variable light domains. It should be noted that this VL canbe paired with any of the other variable heavy domains depicted herein.

FIGS. 21A and 21B depict the sequence for illustrativeaffinity-optimized 1A7 VH/VH pairs. It should be noted that these pairsmay be formatted as Fabs or as scFvs.

FIG. 22 depicts illustrative affinity-engineered 1A7 VH/VL pairs andtheir binding affinities in the context of A) scFvs (in the context of1+1 Fab-scFv-Fc bsAb format) and B) Fab (in the context of 2+1 CLC bsAbformat).

FIG. 23 depicts the sequences for XENP27181, a bivalent anti-CD28 mAbbased on HuTN228 binding domain and IgG1 backbone withE233P/L234V/L235A/G236del/S267K ablation variant; and XENP27656, amonovalent anti-CD28 mAb based on HuTN228 binding domain (formatted asan scFv) and IgG1 backbone with E233P/L234V/L235A/G236del/S267K ablationvariant. CDRs are underlined and slashes indicate the border(s) betweenthe variable regions and constant domain. As noted herein and is truefor every sequence herein containing CDRs, the exact identification ofthe CDR locations may be slightly different depending on the numberingused as is shown in Table 2, and thus included herein are not only theCDRs that are underlined but also CDRs included within the V_(H) andV_(L) domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 24 depicts K_(Dapp) (K_(D) apparent due to bivalent binding) ofvarious CD28 binding phage clones (formatted as bivalent mAbs) for humanCD28 as determined by Octet. First 60 seconds of dissociation was usedfor data fit.

FIG. 25 depicts binding of illustrative bivalent anti-CD28 mAbs based onphage-derived clones on human PBMCs. The data show that the phagecampaign generated CD28 binding domains having weaker maximum bindingthan prior art HuTN228 (which is related to the humanized CD28 bindingdomains described in Example 1A).

FIG. 26 depicts the variable heavy and variable light chain sequencesfor 2E4A3.189, an exemplary phage-derived B7H3 binding domain, as wellas the sequences for XENP32637, an anti-B7H3 mAb based on 2E4A3.189 andIgG1 backbone with E233P/L234V/L235A/G236del/S267K ablation variant.CDRs are underlined and slashes indicate the border(s) between thevariable regions and constant domain. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 2, and thus included herein are not only the CDRsthat are underlined but also CDRs included within the V_(H) and V_(L)domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 27 depicts the sequence for affinity-optimized variable heavy2E4A3.189_H1.22. It should be noted that this VH can be paired with anyof the other variable light domains (VL) depicted herein.

FIG. 28 depicts the variable heavy and variable light chain sequencesfor humanized 6A1, an exemplary rat hybridoma-derived B7H3 bindingdomain, as well as the sequences for XENP33383, an anti-B7H3 mAb basedon 6A1 and IgG1 backbone with E233P/L234V/L235A/G236del/S267K ablationvariant. CDRs are underlined and slashes indicate the border(s) betweenthe variable regions and constant domain. As noted herein and is truefor every sequence herein containing CDRs, the exact identification ofthe CDR locations may be slightly different depending on the numberingused as is shown in Table 2, and thus included herein are not only theCDRs that are underlined but also CDRs included within the V_(H) andV_(L) domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 29 depicts the variable heavy and variable light chain sequencesfor humanized 3C4, an exemplary rat hybridoma-derived B7H3 bindingdomain. CDRs are underlined and slashes indicate the border(s) betweenthe variable regions and constant domain. As noted herein and is truefor every sequence herein containing CDRs, the exact identification ofthe CDR locations may be slightly different depending on the numberingused as is shown in Table 2, and thus included herein are not only theCDRs that are underlined but also CDRs included within the V_(H) andV_(L) domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 30 depicts the variable heavy and variable light chain sequencesfor humanized 4F12, an exemplary rabbit hybridoma-derived B7H3 bindingdomain. CDRs are underlined and slashes indicate the border(s) betweenthe variable regions and constant domain. As noted herein and is truefor every sequence herein containing CDRs, the exact identification ofthe CDR locations may be slightly different depending on the numberingused as is shown in Table 2, and thus included herein are not only theCDRs that are underlined but also CDRs included within the V_(H) andV_(L) domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 31 depicts the variable heavy and variable light chain sequencesfor humanized 38E2, an exemplary rabbit hybridoma-derived B7H3 bindingdomain. CDRs are underlined and slashes indicate the border(s) betweenthe variable regions and constant domain. As noted herein and is truefor every sequence herein containing CDRs, the exact identification ofthe CDR locations may be slightly different depending on the numberingused as is shown in Table 2, and thus included herein are not only theCDRs that are underlined but also CDRs included within the V_(H) andV_(L) domains using other numbering systems. Furthermore, as for all thesequences in the Figures, these V_(H) and V_(L) sequences can be usedeither in a scFv format or in a Fab format.

FIG. 32 depicts the monovalent binding affinities (K_(D)) of variousB7H3 binding domains in the context of 1+1 bispecific formats. It shouldbe noted that the 2E4A3_H1.22_1A7_L1 and 2E4A3_H1.3_1A7_L1 utilize theV_(L) of anti-CD28 clone 1A7.

FIGS. 33A-33E depict exemplary formats of the present invention. FIG.33A depicts the “1+1 Fab-scFv-Fc” format, with a first Fab arm binding afirst antigen and a second scFv arm binding second antigen. The 1+1Fab-scFv-Fc format comprises a first monomer comprising a first heavychain variable region (VH1) covalently attached to the N-terminus of afirst heterodimeric Fc backbone (optionally via a linker), a secondmonomer comprising a single-chain Fv covalently attached to theN-terminus of a second corresponding heterodimeric Fc backbone(optionally via a linker), and a third monomer comprising a light chainvariable region covalently to a light chain constant domain, wherein thelight chain variable region is complementary to the VH1. FIG. 33Bdepicts the “2+1 Fab₂-scFv-Fc” format, with a first Fab arm and a secondFab-scFv arm, wherein the Fab binds a first antigen and the scFv bindssecond antigen. The 2+1 Fab₂-scFv-Fc format comprises a first monomercomprising a first heavy chain variable region (VH1) covalently attachedto the N-terminus of a first heterodimeric Fc backbone (optionally via alinker), a second monomer comprising the VH1 covalently attached(optionally via a linker) to a single-chain Fv covalently attached(optionally via a linker) to the N-terminus of a second correspondingheterodimeric Fc backbone, and a third monomer comprising a light chainvariable region covalently to a light chain constant domain, wherein thelight chain variable region is complementary to the VH1. FIG. 33Cdepicts the “1+1 Common Light Chain” or “1+1 CLC” format, with a firstFc comprising a first Fab arm binding a first antigen and a second Fccomprising a second Fab arm binding second antigen. The 1+1 CLC formatcomprises a first monomer comprising VH1-CH1-hinge-CH2-CH3, a secondmonomer comprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprisingVL-CL. The VL pairs with the VH1 to form a binding domain with a firstantigen binding specificity; and the VL pairs with the VH2 to form abinding domain with a second antigen binding specificity. FIG. 33Ddepicts the “2+1 Common Light Chain” or “2+1 CLC” format, with a firstFc comprising 2 Fab arms each binding a first antigen and a second Fccomprising 1 Fab arm binding a second antigen. The 2+1 CLC formatcomprises a first monomer comprisingVH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomer comprisingVH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VLpairs with the first and second VH1 to form binding domains with a firstantigen binding specificity; and the VL pairs with the VH2 to form abinding domain with a second antigen binding specificity.

FIG. 33E depicts the “2+1 mAb-scFv” format, with a first Fc comprisingan N-terminal Fab arm binding a first antigen and a second Fc comprisingan N-terminal Fab arm binding the first antigen and a C-terminal scFvbinding a second antigen. The 2+1 mAb-scFv format comprises a firstmonomer comprising VH1-CH1-hinge-CH2-CH3, a second monomer comprisingVH1-CH1-hinge-CH2-CH3-scFv, and a third monomer comprising VL-CL. The VLpairs with the first and second VH1 to form binding domains with bindingspecificity for the first antigen.

FIGS. 34A-34E depict exemplary formats of the present invention asutilized in CD28 bispecific antibodies. FIG. 34A depicts the “1+1Fab-scFv-Fc” format, with a first Fab arm binding a tumor-associatedantigen and a second scFv arm binding CD28. The 1+1 Fab-scFv-Fc formatcomprises a first monomer comprising a first heavy chain variable region(VH1) covalently attached to the N-terminus of a first heterodimeric Fcbackbone (optionally via a linker), a second monomer comprising asingle-chain Fv covalently attached to the N-terminus of a secondcorresponding heterodimeric Fc backbone (optionally via a linker), and athird monomer comprising a light chain variable region covalently to alight chain constant domain, wherein the light chain variable region iscomplementary to the VH1.

FIG. 34B depicts the “2+1 Fab₂-scFv-Fc” format, with a first Fab arm anda second Fab-scFv arm, wherein the Fab binds a tumor-associated antigenand the scFv binds CD28. The 2+1 Fab₂-scFv-Fc format comprises a firstmonomer comprising a first heavy chain variable region (VH1) covalentlyattached to the N-terminus of a first heterodimeric Fc backbone(optionally via a linker), a second monomer comprising the VH1covalently attached (optionally via a linker) to a single-chain Fvcovalently attached (optionally via a linker) to the N-terminus of asecond corresponding heterodimeric Fc backbone, and a third monomercomprising a light chain variable region covalently to a light chainconstant domain, wherein the light chain variable region iscomplementary to the VH1. FIG. 34C depicts the “1+1 Common Light Chain”or “1+1 CLC” format, with a first Fc comprising a first Fab arm bindinga tumor-associated antigen and a second Fc comprising a second Fab armbinding CD28. The 1+1 CLC format comprises a first monomer comprisingVH1-CH1-hinge-CH2-CH3, a second monomer comprisingVH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VLpairs with the VH1 to form a binding domain with a first antigen bindingspecificity; and the V_(L) pairs with the VH2 to form a binding domainwith a second antigen binding specificity. FIG. 34D depicts the “2+1Common Light Chain” or “2+1 CLC” format, with a first Fc comprising 2Fab arms each binding a tumor-associated antigen and a second Fccomprising 1 Fab arm binding CD28. The 2+1 CLC format comprises a firstmonomer comprising VH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomercomprising VH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL.The VL pairs with the first and second VH1 to form binding domains witha first antigen binding specificity; and the VL pairs with the VH2 toform a binding domain with a second antigen binding specificity. FIG.34E depicts the “2+1 mAb-scFv” format, with a first Fc comprising anN-terminal Fab arm binding a tumor-associated antigen and a second Fccomprising an N-terminal Fab arm binding a tumor-associated antigen anda C-terminal scFv binding CD28. The 2+1 mAb-scFv format comprises afirst monomer comprising VH1-CH1-hinge-CH2-CH3, a second monomercomprising VH1-CH1-hinge-CH2-CH3-scFv, and a third monomer comprisingVL-CL. The V_(L) pairs with the first and second VH1 to form bindingdomains with binding specificity for the tumor-associated antigen.

FIGS. 35A and 35B depict the sequences for illustrative αB7H3ΔαCD28bsAbs in the 1+1 Fab-scFv-Fc format. CDRs are underlined and slashesindicate the border(s) between the variable regions, linkers, Fcregions, and constant domains. It should be noted that the αB7H3ΔαCD28bsAbs can utilize variable region, Fc region, and constant domainsequences that are 90, 95, 98 and 99% identical (as defined herein),and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 36A-36C depict the sequences for illustrative αB7H3ΔαCD28 bsAbs inthe 2+1 Fab₂-scFv-Fc format. CDRs are underlined and slashes indicatethe border(s) between the variable regions, linkers, Fc regions, andconstant domains. The scFv domain has orientation (N- to C-terminus) ofV_(H)-scFv linker-V_(L), although this can be reversed. It should benoted that the scFv domain sequences includes as the scFv linker betweenthe variable heavy and variable light region the sequenceGKPGSGKPGSGKPGSGKPGS (SEQ ID NO:796); however, this linker can bereplaced with any of the scFv linkers in FIG. 6 . It should also benoted that the Chain 2 sequences include as the domain linker betweenthe C-terminus of the scFv and the N-terminus of the CH2 domain thesequence GGGGSGGGGSKTHTCPPCP (SEQ ID NO:818), which is a “flex halfhinge” domain linker; however, this linker can be replaced with any ofthe “useful domain linkers” of FIG. 7 . It should be noted that theαB7H3ΔαCD28 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 37 depicts the sequences for illustrative αB7H3ΔαCD28 bsAbs in the1+1 CLC format. CDRs are underlined and slashes indicate the border(s)between the variable regions, linkers, Fc regions, and constant domains.It should be noted that the αB7H3×αCD28 bsAbs can utilize variableregion, Fc region, and constant domain sequences that are 90, 95, 98 and99% identical (as defined herein), and/or contain from 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 amino acid substitutions. In addition, each sequenceoutlined herein can include or exclude the M428L/N434S variants in oneor preferably both Fc domains, which results in longer half-life inserum.

FIGS. 38A-38E depicts the sequences for illustrative αB7H3ΔαCD28 bsAbsin the 2+1 CLC format. CDRs are underlined and slashes indicate theborder(s) between the variable regions, linkers, Fc regions, andconstant domains. The scFv domain has orientation (N- to C-terminus) ofV_(H)-scFv linker-V_(L), although this can be reversed. It should benoted that the Chain 2 sequences include as a domain linker (doubleunderlined) the sequence EPKSCGKPGSGKPGS (SEQ ID NO:1182); however, thislinker can be replaced with any domain linker include any of the “usefuldomain linkers” of FIG. 6 . It should be noted that the αB7H3ΔαCD28bsAbs can utilize variable region, Fc region, and constant domainsequences that are 90, 95, 98 and 99% identical (as defined herein),and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIGS. 39A-39E depict the sequences for illustrative αB7H3ΔαCD28 bsAbs inthe 2+1 mAb-scFv format. CDRs are underlined and slashes indicate theborder(s) between the variable regions, linkers, Fc regions, andconstant domains. The scFv domain has orientation (N- to C-terminus) ofV_(H)-scFv linker-V_(L), although this can be reversed. It should benoted that the Chain 2 sequences include as a domain linker the sequenceGKPGSGKPGSGKPGSGKPGS (SEQ ID NO:796); however, this linker can bereplaced with any domain linker include any of the “useful domainlinkers” of FIG. 6 . It should be noted that the αB7H3ΔαCD28 bsAbs canutilize variable region, Fc region, and constant domain sequences thatare 90, 95, 98 and 99% identical (as defined herein), and/or containfrom 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. Inaddition, each sequence outlined herein can include or exclude theM428L/N434S variants in one or preferably both Fc domains, which resultsin longer half-life in serum.

FIGS. 40A and 40B depict A) classic T cell/APC interaction and B)replication of the classic T cell/APC interaction by combining CD3bispecific antibodies with CD28 bispecific antibodies. In classic Tcell/APC interaction, there is a first signal provided by TCR reactivitywith peptide-MHC (Signal 1) and a second signal provided by CD28crosslinking by CD80/CD86 being expressed on APCs (Signal 2) whichtogether fully activate T cells. In contrast in treatment with CD3bispecifics, only the first signal is provided. The CD28 signal may beprovided by a CD28 bispecific with the idea to promote activation andproliferation through CD28 costimulation.

FIG. 41 depicts the introduction of CD28 signaling by a CD28 bispecificantibody and mitigation of any checkpoint mediated repression of theadded CD28 signal by checkpoint blockade (e.g. PD-1 blockade).

FIG. 42 depicts induction of IL-2 release by effector cells in thepresence of MCF7 cancer cells transfected with anti-CD3 scFv (1:1effector:target ratio) and B7H3×CD3 bsAbs XENP34339, XENP35612,XENP35611, and XENP34336 (respectively having CD28 binding affinities of77 nM, 270 nM, 610 nM, and 440 nM). The data show that reducing CD28binding affinity reduces potency of the B7H3×CD28 bispecific antibodies.

FIGS. 43A-43C depict induction of IL-2 secretion from T cells byB7H3×CD28 bsAbs in the presence of A) MDA-MB-231, B) LnCAP, and C) DU145target cells (1:1 E:T ratio) and a constant dose of a illustrativeB7H3×CD3 bsAb.

FIG. 44 depicts consensus framework regions (FR) and complementaritydetermining regions (CDRs) (as in Kabat) for anti-CD28 clone 1A7variable heavy and variable light domain variants.

FIGS. 45A-45D depict the pharmacokinetics of B7H3×CD28 bsAbs in variousantibody formats in a cynomolgus study. The data show that at each doselevel investigated, the 2+1 common light chain format had the besthalf-life and pharmacokinetics.

FIGS. 46A-46H depict the change in serum concentration level over timein cynomolgus monkeys dosed with A) XENP34398, B) XENP37808, C)XENP37810, D) XENP34732, E) XENP35151, F) XENP351535, G) XENP37807, andH) XENP37982. Relative doses are depicted as follows: circle for 0.5×dose; upside down triangle for 1.3× dose; hexagon for 1.8× dose; squarefor 2× dose; diamond for 3.25× dose; star for 4.5× dose; and trianglefor 5× dose.

FIG. 47 summarizes properties of B7H3×CD28 bsAbs XENP34398, XENP37808,XENP34732, and XENP35153. It should be noted that some of the datadepicted in this summary table may not be the same experimental datadepicted elsewhere in the Working Examples as some of those illustrateexperimental data from earlier stages of development.

FIGS. 48A and 48B depict IFNγ release following incubation of A) A549cancer cells and B) SKOV-3 cancer cells with CD3⁺ T cells (10:1effector:target ratio) and indicated concentration of B7H3×CD28bispecific antibodies XENP34339 or XENP34717. The data show that bothXENP34339 and XENP34717 induced cytokine release by the T cells.XENP34339 having bivalent B7H3 binding induced cytokine release morepotently than XENP34717 having monovalent B7H3 binding.

FIG. 49 depicts the restoration of CD28 signaling in a mixed lymphocytereaction (following incubation of with 1 μg/mL CTLA-4-Fc) by XENP34339.Error bars represent the mean expression in culture supernatants fromone MLR reaction tested in technical quadruplicate.

FIGS. 50A and 50B depict IFNγ release following incubation of NLV-loadedMDA-MB-231 cancer cells with CD3⁺ T cells purified from A) a first donorand B) a second donor at a 10:1 effector:target ratio and the indicatedcombinations of XENP16432, XENP34339, and XENP34389. The data show thatincubation with XENP34339 alone induced cytokine release from T cellsand combined synergistically with PD-1 blockade to enhance cytokinerelease.

FIG. 51 depicts expansion of NLV-tetramer positive cells followingincubation of NLV-loaded MCF7 cancer cells with purified CD3⁺ T cellspurified at a 10:1 effector:target ratio and the indicated combinationsof XENP16432 and XENP34339. The data show that combination of XENP34339with PD-1 blockade enhanced expansion of NLV-tetramer positive CD8⁺ Tcells.

FIGS. 52A and 52B depicts the dissociation constant (K_(D); andcorresponding sensorgrams) of anti-B7H3 clone 2E4A3.189 and clone 6A1for either the full B7H3 extracellular V1C1-V2V2 domain or theindividual V1C1 or V2C2 domains.

FIG. 53 depicts the dissociation constant (K_(D); and correspondingsensorgrams) of anti-CD28 clone 1A7 affinity variant H1.14_L1 as a Fabin the 2+1 CLC format or as an scFv in the 2+1 Fab₂-scFv-Fc format forCD28 antigen.

FIGS. 54A and 54B depict the sequences for illustrative αPSMA×αCD3 bsAbsin the 2+1 Fab₂-scFv-Fc format and comprising a H1.30 L1.47 anti-CD3scFv (a.k.a. CD3 High [VHVL]). CDRs are underlined and slashes indicatethe border(s) between the variable regions and other chain components(e.g. constant region and domain linkers). It should be noted that theαPSMA×αCD3 bsAbs can utilize variable region, Fc region, and constantdomain sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions. In addition, each sequence outlined herein can include orexclude the M428L/N434S variants in one or preferably both Fc domains,which results in longer half-life in serum.

FIG. 55 depicts cell kill over time following incubation of LNCaP cancercells (PSMA⁺B7H3⁺) with CD3⁺ T cells at a 1:1 effector:target ratio andillustrative CD3 bispecific (αPSMA×αCD3 XENP31602) alone or incombination with XENPXENP34339 at the indicated concentrations. The datashow that XENP31602 αPSMA×αCD3 alone minimally enhanced cell kill incomparison to incubation of cancer and T cells alone. Addition ofXENP34339 αB7H3ΔαCD28 overcomes cancer cell resistance to the CD3bispecific.

FIGS. 56A-56L depict sequences for exemplary anti-CD3 binding domainssuitable for use in CD3 bispecific antibodies which may be combined withthe CD28 bispecific antibodies of the invention. The CDRs areunderlined, the scFv linker is double underlined (in the sequences, thescFv linker is a positively charged scFv (GKPGS)₄ linker (SEQ ID NO:796), although as will be appreciated by those in the art, this linkercan be replaced by other linkers, including uncharged or negativelycharged linkers, some of which are depicted in FIG. 6 ), and the slashesindicate the border(s) of the variable domains. In addition, the namingconvention illustrates the orientation of the scFv from N- toC-terminus. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in Table2, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the V_(H) and V_(L) domains using othernumbering systems. Furthermore, as for all the sequences in the Figures,these V_(H) and V_(L) sequences can be used either in a scFv format orin a Fab format.

FIGS. 57A-57C depict A) IFNγ release, B) IL-2 release, and C) CD3⁺ Tcell expansion following incubation of LNCaP cancer cells (PSMA⁺B7H3⁺)with CD3⁺ T cells at a 1:1 effector:target ratio and 1 μg/ml XENP34339in combination with a dose titration of an illustrative CD3 bispecific(αPSMA×αCD3 XENP31602).

FIGS. 58A-58C depict A) IFNγ release, B) IL-2 release, and C) CD3⁺ Tcell expansion following incubation of 22Rv1 cancer cells (PSMA⁺B7H3⁺)with CD3⁺ T cells at a 1:1 effector:target ratio and 1 μg/ml XENP34339in combination with a dose titration of an illustrative CD3 bispecific(αPSMA×αCD3 XENP31602).

FIGS. 59A-59C depict A) IFNγ release, B) IL-2 release, and C) CD3⁺ Tcell expansion following incubation of SKOV-3 cancer cells (PSMA⁻B7H3⁺)with CD3⁺ T cells at a 1:1 effector:target ratio and 1 μg/ml XENP34339in combination with a dose titration of an illustrative CD3 bispecific(αPSMA×αCD3 XENP31602).

FIGS. 60A-60C depict A) IFNγ release, B) 1L-2 release, and C) CD3⁺ Tcell expansion following incubation of OVCAR-8 cancer cells (PSMA⁻B7H3⁺)with CD3⁺ T cells at a 1:1 effector:target ratio and 1 μg/ml XENP34339in combination with a dose titration of an illustrative CD3 bispecific(αPSMA×αCD3 XENP31602).

FIGS. 61A-61E depict change in tumor volume (as determined by calipermeasurement; baseline corrected) in individual mouse over time (in days)in pp65-MDA-MB-231 and huPBMC-engrafted NSG mice dosed with A) a firstillustrative B7H3×CD3 bispecific antibody (CD3bsAb1) (0.5 mg/kg) alone,B) a second illustrative B7H3×CD3 bispecific antibody (CD3bsAb2) (0.5mg/kg) alone, C) a combination of XENP34339 (5.0 mg/kg) with CD3bsAb1(0.5 mg/kg), D) a combination of XENP34339 (5.0 mg/kg) with CD3bsAb2(0.5 mg/kg), or E) PBS. F) depicts

FIG. 62 depicts group median change in tumor volume (as determined bycaliper measurement; baseline corrected) over time (in days) inpp65-MDA-MB-231 and huPBMC-engrafted NSG mice dosed with a firstillustrative B7H3×CD3 bispecific antibody (CD3bsAb1) (0.5 mg/kg) alone,a second illustrative B7H3×CD3 bispecific antibody (CD3bsAb2) (0.5mg/kg) alone, a combination of XENP34339 (5.0 mg/kg) with CD3bsAb1 (0.5mg/kg), a combination of XENP34339 (5.0 mg/kg) with CD3bsAb2 (0.5mg/kg), or PBS control.

FIG. 63 depicts CD45+ cell counts in blood of pp65-MDA-MB-231 andhuPBMC-engrafted NSG mice dosed with a first illustrative B7H3×CD3bispecific antibody (CD3bsAb1) (0.5 mg/kg) alone, a second illustrativeB7H3×CD3 bispecific antibody (CD3bsAb2) (0.5 mg/kg) alone, a combinationof XENP34339 (5.0 mg/kg) with CD3bsAb1 (0.5 mg/kg), a combination ofXENP34339 (5.0 mg/kg) with CD3bsAb2 (0.5 mg/kg), or PBS control on Day14 after first dose.

FIGS. 64A-64E depict expansion of A) CD45+, B) CD4+(all), C) CD8+(all),D) CD4+(Ki67+), and E) CD8+(Ki67+) cells in blood (as indicated bycount) of 22RV1 and huPBMC-engrafted NSG-DKO mice dosed with a low orhigh concentration doses of illustrative PSMA×CD3 bsAb XENP32220 aloneor in combination with XENP34339. Treatment with both CD3 and CD28 bsAbsenhanced T cell expansion in comparison to treatment with CD3 bsAbalone.

FIGS. 65A-65D depicts A) activation of CD4+ cells (as indicated by CD25expression), B) activation of CD4+ cells (as indicated by PD1expression), C) activation of CD8+ cells (as indicated by CD25expression), and D) activation of CD8+ cells (as indicated by PD1expression) in blood (as indicated by count) of 22RV1 andhuPBMC-engrafted NSG-DKO mice dosed with a low or high concentrationdoses of illustrative PSMA×CD3 bsAb XENP32220 alone or in combinationwith XENP34339. Treatment with both CD3 and CD28 bsAbs enhanced T cellactivation in comparison to treatment with CD3 bsAb alone.

FIG. 66 depicts group median change in tumor volume (as determined bycaliper measurement; baseline corrected) over time (in days) inpp65-MDA-MB-231-engrafted CD34+ Hu-NSG mice dosed with an illustrativeB7H3×CD3 bispecific antibody (0.5 mg/kg) alone, XENP35612 alone (1mg/kg) alone, a combination of XENP34339 (0.3 mg/kg) with the B7H3×CD3bsAb (0.5 mg/kg), a combination of XENP35612 (1 mg/kg) with the B7H3×CD3bsAb (0.5 mg/kg), or PBS control.

FIGS. 67A and 67B depict baseline corrected tumor volume on A) Day 6 andB) Day 9 (post-dose) in pp65-MDA-MB-231-engrafted CD34+ Hu-NSG micedosed with a illustrative B7H3×CD3 bispecific antibody (0.5 mg/kg)alone, XENP35612 alone (1 mg/kg) alone, a combination of XENP34339 (0.3mg/kg) with the B7H3×CD3 bsAb (0.5 mg/kg), a combination of XENP35612 (1mg/kg) with the B7H3×CD3 bsAb (0.5 mg/kg), or PBS control. Statisticsperformed on baseline corrected data using Mann-Whitney test.

FIGS. 68A and 68B depict expansion of A) CD45+ and B) CD8+ cells intumor of pp65-MDA-MB-231-engrafted CD34+ Hu-NSG mice dosed with anillustrative B7H3×CD3 bispecific antibody (0.5 mg/kg) alone, XENP35612alone (1 mg/kg) alone, a combination of XENP35612 (1 mg/kg) with theB7H3×CD3 bsAb (0.5 mg/kg), or PBS control. Statistics performed onlog-transformed data using unpaired t-test.

FIG. 69 depicts the sequences for XENP29154, which is in-house producedTGN1412.

FIGS. 70A-70C depict the release of A) IFNγ, B) IL-6, and C) TNFα fromhuman PBMCs treated with air-dried XENP34339, TGN1412 (XENP29154), ornegative control PBS.

FIGS. 71A-71C depict the release of A) IFNγ, B) IL-2, and C) TNFα fromhuman PBMCs treated with air-dried XENP37808, TGN1412 (XENP29154), ornegative control PBS.

FIGS. 72A and 72B depict induction of IL-2 release by A) PBMCs from ahuman donor or B) PBMCs from a cynomolgus donor by XENP37808 in thepresence of HEK cells transfected with αCD3 scFv (with or without B7H3knockout).

FIGS. 73A and 73B depict induction of RTCC on A) 22RV-NLR (having ˜170KB7H3 antigen density) and B) DU145-NLR (having ˜270K B7H3 antigendensity) target cells B7H33×CD3 mAb alone, or in combination with eitherXENP34398 or XENP37808. The data show that XENP34398 and XENP37808 (incombination with B7H3 X CD3) induce very similar levels of RTCC.

FIGS. 74A-74C depict induction of IL-2 release by T cells in thepresence of A) OVCAR8 (having ˜20K B7H3 antigen density), B) 22RV1-NLR(having ˜170K B7H3 antigen density), and C) DU145-NLR (having ˜270K B7H3antigen density), and XENP34398 or XENP37808 in combination with aB7H3×CD3 bsAb.

FIG. 75 depicts consensus framework regions (FR) and complementaritydetermining regions (CDRs) (as in Kabat) for anti-B7H3 clone 2E4A3.189variable heavy and variable light domain variants.

DETAILED DESCRIPTION I. Overview

The activation of T cells in the treatment of cancer is being widelyinvestigated. T cells require multiple signals for complete activationand differentiation. As shown in FIG. 40 , Signal 1, promoted byrecognition of a peptide-MHC (pMHC) complex by the T cell receptor(TCR), is absolutely required for T cell activation. Signal 2, whichsynergizes with, and amplifies signal 1, is typically provided by theinteraction of the CD28 ligands CD80 and CD86 with CD28 itself. AlthoughCD28 engagement alone is typically inert, when combined with signal 1activation, it promotes additional activation, survival, andproliferative signals, including IL2 secretion. As CD80 and CD86 areonly naturally expressed by professional antigen-presenting cells (APC),the extent of CD28 costimulation in the tumor setting can be highlyvariable. Accordingly, the present invention is directed to a novelclass of tumor-targeted CD28 bispecific antibodies (including B7H3×CD28more fully described herein), the CD80/CD86 engagement of CD28 can bemimicked, providing an artificial source of signal 2. Notably, signalcan either be provided by the natural TCR:pMHC recognition of tumorcells, or it can be provided by combination of the CD28 bispecific witha CD3 bispecific (which can mimick signal 1).

Accordingly, provided herein are novel anti-CD28×anti-B7H3 (alsoreferred to as “αCD28×αB7H3” and sometimes “CD28×B7H3”) bispecificantibodies and methods of using such antibodies for the treatment ofcancers. In many cases, these bispecific antibodies are heterodimeric.Subject αCD28×αB7H3 antibodies are capable of agonistically binding toCD28 costimulatory molecules on T cells and targeting to B7H3 on tumorcells. Thus, such antibodies selectively enhance anti-tumor activity attumor sites while minimizing peripheral toxicity. The subject antibodiesprovided herein are particularly useful for enhancing anti-tumoractivity either alone, as a monotherapy, or when used in combinationwith other anti-cancer therapies as more fully described herein

Accordingly, in one aspect, provided herein are heterodimeric antibodiesthat bind to two different antigens, e.g., the antibodies are“bispecific,” in that they bind two different target antigens, generallyCD28 and B7H3 as described below. These heterodimeric antibodies canbind each of the target antigens either monovalently (e.g., there is asingle antigen binding domain such as a variable heavy and variablelight domain pair) or bivalently (there are two antigen binding domainsthat each independently bind the antigen). In some embodiments, theheterodimeric antibody provided herein includes one CD28 binding domainand one B7H3 binding domain (e.g., heterodimeric antibodies in the “1+1Fab-scFv-Fc” format described herein, which are thus bispecific andbivalent). In other embodiments, the heterodimeric antibody providedherein includes one CD28 binding domain and two B7H3 binding domains(e.g., heterodimeric antibodies in the “2+1 Fab₂-scFv-Fc” formatsdescribed herein, which are thus bispecific but trivalent, as theycontain three antigen binding domains (ABDs)). The heterodimericantibodies provided herein are based on the use of different monomersthat contain amino acid substitutions (i.e., skew variants”) that “skew”formation of heterodimers over homodimers, as is more fully outlinedbelow. In some embodiments, the heterodimer antibodies are also coupledwith “pI variants” that allow simple purification of the heterodimersaway from the homodimers, as is similarly outlined below. Theheterodimeric bispecific antibodies provided generally rely on the useof engineered or variant Fc domains that can self-assemble in productioncells to produce heterodimeric proteins, and methods to generate andpurify such heterodimeric proteins.

II. Nomenclature

The antibodies provided herein are listed in several different formats.In some instances, each monomer of a particular antibody is given aunique “XENP” number, although as will be appreciated in the art, alonger sequence might contain a shorter one. For example, a “scFv-Fc”monomer of a 1+1 Fab-scFv-Fc format antibody may have a first XENPnumber, while the scFv domain itself will have a different XENP number.Some molecules have three polypeptides, so the XENP number, with thecomponents, is used as a name. Thus, the molecule XENP34389, which is in2+1 Fab₂-scFv-Fc format, comprises three sequences (see FIG. 28A) a“Fab-Fc Heavy Chain” monomer; 2) a “Fab-scFv-Fc Heavy Chain” monomer;and 3) a “Light Chain” monomer or equivalents, although one of skill inthe art would be able to identify these easily through sequencealignment. These XENP numbers are in the sequence listing as well asidentifiers, and used in the Figures. In addition, one molecule,comprising the three components, gives rise to multiple sequenceidentifiers. For example, the listing of the Fab includes the full heavychain sequence, the variable heavy domain sequence and the three CDRs ofthe variable heavy domain sequence, the full light chain sequence, avariable light domain sequence and the three CDRs of the variable lightdomain sequence. A Fab-scFv-Fc monomer includes a full-length sequence,a variable heavy domain sequence, 3 heavy CDR sequences, and an scFvsequence (include scFv variable heavy domain sequence, scFv variablelight domain sequence and scFv linker). Note that some molecules hereinwith a scFv domain use a single charged scFv linker (+H), althoughothers can be used. In addition, the naming nomenclature of particularantigen binding domains (e.g., B7H3 and CD28 binding domains) use a“Hx.xx_Ly.yy” type of format, with the numbers being unique identifiersto particular variable chain sequences. Thus, the variable domain of theFab side of B7H3 binding domain 6A[B7H3] (e.g., FIG. 28A) is “H1_L1”,which indicates that the variable heavy domain, H1, was combined withthe light domain L1. In the case that these sequences are used as scFvs,the designation “H1_L1”, indicates that the variable heavy domain, H1 iscombined with the light domain, L1, and is in V_(H)-linker-V_(L)orientation, from N- to C-terminus. This molecule with the identicalsequences of the heavy and light variable domains but in the reverseorder (V_(L)-linker-V_(H) orientation, from N- to C-terminus) would bedesignated “L1_H1”. Similarly, different constructs may “mix and match”the heavy and light chains as will be evident from the sequence listingand the figures.

Additionally, with regard to the sequence listing, SEQ ID NOs:1 to 88correspond to antigen binding domains previously shown in FIG. 17 ofU.S. Ser. No. 63/092,272; SEQ ID NOs: 89-496 correspond to antigenbinding domains previously shown in FIG. 24 of U.S. Ser. No. 63/092,272.Additionally, SEQ ID NOs: 497 to 584 are all variant variable heavydomains of the 2E4A3.189[B7H3] parental antibody, all of which find usein the present invention as more fully outlined below. SEQ ID NOs:585 to651 are all variant variable heavy domains of the 1A7[CD28] parentalantibody, all of which find use in the present invention. SEQ ID NOs:652to 756 are all variant variable light domains of the 1A7[CD28] parentalantibody, all of which find use in the present invention.

III. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “CD28,” “Cluster of Differentiation 28,” and “Tp44” (e.g., GenebankAccession Numbers NP_001230006 (human), NP_001230007 (human), NP_006130(human), and NP_031668 (mouse)) herein is meant a B7 receptor expressedon T cells that provides co-stimulatory signals required for T cellactivation and survival. T cell stimulation through CD28 in addition tothe T cell receptor (TCR) provides a potent signal for the production ofvarious interleukins. CD28 is the receptor for CD80 (B7.1) and CD86(B7.2) proteins. CD28 includes an intercellular domain with a YMNM motifcritical for the recruitment of SH2-domain containing proteins,particularly PI3K. CD28 also includes two proline-rich motifs that areable to bind SH3-containing proteins. Exemplary CD28 sequences aredepicted in FIG. 1 . Unless otherwise noted, references to CD28 are tothe human CD28 sequence.

By “B7H3,” “B7-H3,” “B7RP-2,” “CD276,” “Cluster of Differentiation 276,”(e.g., Genebank Accession Numbers NP_001019907 (human), NP_001316557(human), NP_001316558 (human), NP_079516 (human), and NP_598744 (mouse))herein is meant a type-1 transmembrane protein that is a member of theB7 family possessing an ectodomain composed of a single IgV-IgC domainpair. B7H3 is an immune checkpoint molecule and is aberrantlyoverexpressed in many types of cancers. Exemplary B7H3 sequences aredepicted in FIGS. 2A and B. Unless otherwise noted, references to B7H3are to the human B7H3 sequence.

By “ablation” herein is meant a decrease or removal of activity. Thus,for example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with more than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore, SPR or BLI assay. Ofparticular use in the ablation of FcγR binding are those shown in FIG. 5, which generally are added to both monomers.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction, wherein nonspecificcytotoxic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause lysis of the target cell. ADCC is correlatedwith binding to FcγRIIIa; increased binding to FcγRIIIa leads to anincrease in ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecificphagocytic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause phagocytosis of the target cell.

As used herein, the term “antibody” is used generally. Antibodiesprovided herein can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described herein.

Traditional immunoglobulin (Ig) antibodies are “Y” shaped tetramers.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light chain” monomer(typically having a molecular weight of about 25 kDa) and one “heavychain” monomer (typically having a molecular weight of about 50-70 kDa).

Other useful antibody formats include, but are not limited to, the “1+1Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1common light chain” formats provided herein (see, e.g., FIG. 33 ).Additional useful antibody formats include, but are not limited to,“mAb-Fv,” “mAb-scFv,” “central-Fv”, “one armed scFv-mAb,” “scFv-mAb,”“dual scFv,” and “trident” format antibodies, as disclosed inUS20180127501A1, which is incorporated by reference herein, particularlyin pertinent part relating to antibody formats (see, e.g., FIG. 2 ofUS20180127501A1).

Antibody heavy chains typically include a variable heavy (VH) domain,which includes vhCDR1-3, and an Fc domain, which includes a CH2-CH3monomer. In some embodiments, antibody heavy chains include a hinge andCH1 domain. Traditional antibody heavy chains are monomers that areorganized, from N- to C-terminus: VH—CH1-hinge-CH2-CH3. TheCH1-hinge-CH2-CH3 is collectively referred to as the heavy chain“constant domain” or “constant region” of the antibody, of which thereare five different categories or “isotypes”: IgA, IgD, IgG, IgE and IgM.

In some embodiments, the antibodies provided herein include IgG isotypeconstant domains, which has several subclasses, including, but notlimited to IgG1, IgG2, IgG3, and IgG4. In the IgG subclass ofimmunoglobulins, there are several immunoglobulin domains in the heavychain. By “immunoglobulin (Ig) domain” herein is meant a region of animmunoglobulin having a distinct tertiary structure. Of interest in thepresent invention are the heavy chain domains, including, the constantheavy (CH) domains and the hinge domains. In the context of IgGantibodies, the IgG isotypes each have three CH regions. Accordingly,“CH” domains in the context of IgG are as follows: “CH1” refers topositions 118-215 according to the EU index as in Kabat. “Hinge” refersto positions 216-230 according to the EU index as in Kabat. “CH2” refersto positions 231-340 according to the EU index as in Kabat, and “CH3”refers to positions 341-447 according to the EU index as in Kabat. Asshown in Table 1, the exact numbering and placement of the heavy chaindomains can be different among different numbering systems. As shownherein and described below, the pI variants can be in one or more of theCH regions, as well as the hinge region, discussed below.

It should be noted that IgG1 has different allotypes with polymorphismsat 356 (D or E) and 358 (L or M). The sequences depicted herein use the356E/358M allotype, however the other allotype is included herein. Thatis, any sequence inclusive of an IgG1 Fc domain included herein can have356D/358L replacing the 356E/358M allotype. It should be understood thattherapeutic antibodies can also comprise hybrids of isotypes and/orsubclasses. For example, as shown in US Publication 2009/0163699,incorporated by reference, the present antibodies, in some embodiments,include human IgG1/G2 hybrids.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody, in someinstances, excluding all of the first constant region immunoglobulindomain (e.g., CH1) or a portion thereof, and in some cases, optionallyincluding all or part of the hinge. For IgG, the Fc domain comprisesimmunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all ora portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, insome cases, the Fc domain includes, from N- to C-terminal, CH2-CH3 andhinge-CH2-CH3. In some embodiments, the Fc domain is that from IgG1,IgG2, IgG3 or IgG4, with IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3finding particular use in many embodiments. Additionally, in the case ofhuman IgG1 Fc domains, the hinge may include a C220S amino acidsubstitution. Furthermore, in the case of human IgG4 Fc domains, thehinge may include a S228P amino acid substitution. Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to include residues E216, C226, or A231 to itscarboxyl-terminal, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR or to the FcRn.

By “heavy chain constant region” herein is meant the CH1-hinge-CH2-CH3portion of an antibody (or fragments thereof), excluding the variableheavy domain; in EU numbering of human IgG1 this is amino acids 118-447.By “heavy chain constant region fragment” herein is meant a heavy chainconstant region that contains fewer amino acids from either or both ofthe N- and C-termini but still retains the ability to form a dimer withanother heavy chain constant region.

Another type of domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “hinge domain”herein is meant the flexible polypeptide comprising the amino acidsbetween the first and second constant domains of an antibody.Structurally, the IgG CH1 domain ends at EU position 215, and the IgGCH2 domain begins at residue EU position 231. Thus for IgG the antibodyhinge is herein defined to include positions 216 (E216 in IgG1) to 230(P230 in IgG1), wherein the numbering is according to the EU index as inKabat. In some cases, a “hinge fragment” is used, which contains feweramino acids at either or both of the N- and C-termini of the hingedomain. As noted herein, pI variants can be made in the hinge region aswell. Many of the antibodies herein have at least one the cysteines atposition 220 according to EU numbering (hinge region) replaced by aserine. Generally, this modification is on the “scFv monomer” side (when1+1 or 2+1 formats are used) for most of the sequences depicted herein,although it can also be on the “Fab monomer” side, or both, to reducedisulfide formation. Specifically included within the sequences hereinare one or both of these cysteines replaced (C220S).

As will be appreciated by those in the art, the exact numbering andplacement of the heavy chain constant region domains (i.e., CH1, hinge,CH2 and CH3 domains) can be different among different numbering systems.A useful comparison of heavy constant region numbering according to EUand Kabat is as below, see Edelman et al., 1969, Proc Natl Acad Sci USA63:78-85 and Kabat et al., 1991, Sequences of Proteins of ImmunologicalInterest, 5th Ed., United States Public Health Service, NationalInstitutes of Health, Bethesda, entirely incorporated by reference.

TABLE 1 EU Kabat Numbering Numbering CH1 118-215 114-223 Hinge 216-230226-243 CH2 231-340 244-360 CH3 341-447 361-478

The antibody light chain generally comprises two domains: the variablelight domain (VL), which includes light chain CDRs vlCDR1-3, and aconstant light chain region (often referred to as CL or CK). Theantibody light chain is typically organized from N- to C-terminus:VL-CL.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen (e.g., B7H3 orCD28) as discussed herein. As is known in the art, these CDRs aregenerally present as a first set of variable heavy CDRs (vhCDRs orVHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), eachcomprising three CDRs: vhCDR1, vhCDR2, vhCDR3 variable heavy CDRs andvlCDR1, vlCDR2 and vlCDR3 vhCDR3 variable light CDRs. The CDRs arepresent in the variable heavy domain (vhCDR1-3) and variable lightdomain (vlCDR1-3). The variable heavy domain and variable light domainfrom an Fv region.

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g., a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1,vhCDR2 and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3). A usefulcomparison of CDR numbering is as below, see Lafranc et al., Dev. Comp.Immunol. 27(1):55-77 (2003):

TABLE 2 Kabat + Chothia IMGT Kabat AbM Chothia Contact Xencor vhCDR126-35 27-38 31-35 26-35 26-32 30-35 27-35 vhCDR2 50-65 56-65 50-65 50-5852-56 47-58 54-61 vhCDR3  95-102 105-117  95-102  95-102  95-102  93-101103-116 vlCDR1 24-34 27-38 24-34 24-34 24-34 30-36 27-38 vlCDR2 50-5656-65 50-56 50-56 50-56 46-55 56-62 vlCDR3 89-97 105-117 89-97 89-9789-97 89-96  97-105

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g., Kabat et al., supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of the antigen binding domains andantibodies. “Epitope” refers to a determinant that interacts with aspecific antigen binding site in the variable region of an antibodymolecule known as a paratope. Epitopes are groupings of molecules suchas amino acids or sugar side chains and usually have specific structuralcharacteristics, as well as specific charge characteristics. A singleantigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

In some embodiments, the six CDRs of the antigen binding domain arecontributed by a variable heavy and a variable light domain. In a “Fab”format, the set of 6 CDRs are contributed by two different polypeptidesequences, the variable heavy domain (vh or VH; containing the vhCDR1,vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containingthe vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domainbeing attached to the N-terminus of the CH1 domain of the heavy chainand the C-terminus of the vl domain being attached to the N-terminus ofthe constant light domain (and thus forming the light chain). In a scFvformat, the vh and vl domains are covalently attached, generally throughthe use of a linker (a “scFv linker”) as outlined herein, into a singlepolypeptide sequence, which can be either (starting from the N-terminus)vh-linker-vl or vl-linker-vh, with the former being generally preferred(including optional domain linkers on each side, depending on the formatused. In general, the C-terminus of the scFv domain is attached to theN-terminus of all or part of the hinge in the second monomer.

By “variable region” or “variable domain” as used herein is meant theregion of an immunoglobulin that comprises one or more Ig domainssubstantially encoded by any of the Vκ, Vλ, and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively, and contains the CDRs that confer antigen specificity.Thus, a “variable heavy domain” pairs with a “variable light domain” toform an antigen binding domain (“ABD”). In addition, each variabledomain comprises three hypervariable regions (“complementary determiningregions,” “CDRs”) (vhCDR1, vhCDR2 and vhCDR3 for the variable heavydomain and vlCDR1, vlCDR2 and vlCDR3 for the variable light domain) andfour framework (FR) regions, arranged from amino-terminus tocarboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

By “Fab” or “Fab region” as used herein is meant the antibody regionthat comprises the VH, CH1, VL, and CL immunoglobulin domains, generallyon two different polypeptide chains (e.g., VH—CH1 on one chain and VL-CLon the other). Fab may refer to this region in isolation, or this regionin the context of a bispecific antibody of the invention. In the contextof a Fab, the Fab comprises an Fv region in addition to the CH1 and CLdomains.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant theantibody region that comprises the V_(L) and V_(H) domains. Fv regionscan be formatted as both Fabs (as discussed above, generally twodifferent polypeptides that also include the constant regions asoutlined above) and single chain Fvs (scFvs), where the v and vh domainsare included in a single peptide, attached generally with a linker asdiscussed herein.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (vh-linker-vl orvl-linker-vh). In the sequences depicted in the sequence listing and inthe figures, the order of the vh and vl domain is indicated in the name,e.g., H.X_L.Y means N- to C-terminal is vh-linker-vl, and L.Y_H.X isvl-linker-vh.

Some embodiments of the subject antibodies provided herein comprise atleast one scFv domain, which, while not naturally occurring, generallyincludes a variable heavy domain and a variable light domain, linkedtogether by a scFv linker. As outlined herein, while the scFv domain isgenerally from N- to C-terminus oriented as VH-scFv linker-VL, this canbe reversed for any of the scFv domains (or those constructed using vhand vl sequences from Fabs), to VL-scFv linker-VH, with optional linkersat one or both ends depending on the format.

By “modification” or “variant” herein is meant an amino acidsubstitution, insertion, and/or deletion in a polypeptide sequence or analteration to a moiety chemically linked to a protein. For example, amodification may be an altered carbohydrate or PEG structure attached toa protein. By “amino acid modification” herein is meant an amino acidsubstitution, insertion, and/or deletion in a polypeptide sequence. Forclarity, unless otherwise noted, the amino acid modification is alwaysto an amino acid coded for by DNA, e.g., the 20 amino acids that havecodons in DNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution;” that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233 #, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233- or EDA233 # designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. The protein variant has atleast one amino acid modification compared to the parent protein, yetnot so many that the variant protein will not align with the parentalprotein using an alignment program such as that described below. Ingeneral, variant proteins (such as variant Fc domains, etc., outlinedherein, are generally at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99% identical to the parent protein, using the alignmentprograms described below, such as BLAST.

“Variant” as used herein also refers to particular amino acidmodifications that confer particular function (e.g., a“heterodimerization variant,” “pI variant,” “ablation variant,” etc.).

As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe heavy constant domain or Fc region from IgG1, IgG2, IgG3 or IgG4,although human sequences with variants can also serve as “parentpolypeptides”, for example the IgG1/2 hybrid of US Publication2006/0134105 can be included. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Accordingly, by “antibodyvariant” or “variant antibody” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification, “IgG variant” or “variant IgG” as used herein is meant anantibody that differs from a parent IgG (again, in many cases, from ahuman IgG sequence) by virtue of at least one amino acid modification,and “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain ascompared to an Fc domain of human IgG1, IgG2 or IgG4.

“Fc variant” or “variant Fc” as used herein is meant a proteincomprising an amino acid modification in an Fc domain. The modificationcan be an addition, deletion, or substitution. The Fc variants aredefined according to the amino acid modifications that compose them.Thus, for example, N434S or 434S is an Fc variant with the substitutionfor serine at position 434 relative to the parent Fc polypeptide,wherein the numbering is according to the EU index. Likewise,M428L/N434S defines an Fc variant with the substitutions M428L and N434Srelative to the parent Fc polypeptide. The identity of the WT amino acidmay be unspecified, in which case the aforementioned variant is referredto as 428/434S. It is noted that the order in which substitutions areprovided is arbitrary, that is to say that, for example, 428L/434S isthe same Fc variant as 434S/428L, and so on. For all positions discussedherein that relate to antibodies or derivatives and fragments thereof(e.g., Fc domains), unless otherwise noted, amino acid positionnumbering is according to the EU index. The “EU index” or “EU index asin Kabat” or “EU numbering” scheme refers to the numbering of the EUantibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, herebyentirely incorporated by reference). The modification can be anaddition, deletion, or substitution.

In general, variant Fc domains have at least about 80, 85, 90, 95, 97,98 or 99 percent identity to the corresponding parental human IgG Fcdomain (using the identity algorithms discussed below, with oneembodiment utilizing the BLAST algorithm as is known in the art, usingdefault parameters). Alternatively, the variant Fc domains can have from1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 amino acid modifications as compared to theparental Fc domain. Alternatively, the variant Fc domains can have up to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 amino acid modifications as compared to theparental Fc domain. Additionally, as discussed herein, the variant Fcdomains described herein still retain the ability to form a dimer withanother Fc domain as measured using known techniques as describedherein, such as non-denaturing gel electrophoresis.

By “protein” as used herein is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. In addition, polypeptides that make up the antibodies of theinvention may include synthetic derivatization of one or more sidechains or termini, glycosylation, PEGylation, circular permutation,cyclization, linkers to other molecules, fusion to proteins or proteindomains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the human IgGs comprise a serine at position 434, the substitution434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life. An “FcRn variant” is an amino acidmodification that contributes to increased binding to the FcRn receptor,and suitable FcRn variants are shown below.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Accordingly, by“parent immunoglobulin” as used herein is meant an unmodifiedimmunoglobulin polypeptide that is modified to generate a variant, andby “parent antibody” as used herein is meant an unmodified antibody thatis modified to generate a variant antibody. It should be noted that“parent antibody” includes known commercial, recombinantly producedantibodies as outlined below. In this context, a “parent Fc domain” willbe relative to the recited variant; thus, a “variant human IgG1 Fcdomain” is compared to the parent Fc domain of human IgG1, a “varianthuman IgG4 Fc domain” is compared to the parent Fc domain human IgG4,etc.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for numbering of antibodydomains (e.g., a CH1, CH2, CH3 or hinge domain).

By “target antigen” as used herein is meant the molecule that is boundspecifically by the antigen binding domain comprising the variableregions of a given antibody.

By “strandedness” in the context of the monomers of the heterodimericantibodies of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g., making the pI higher) then steric variants thatare “charge pairs” that can be utilized as well do not interfere withthe pI variants, e.g., the charge variants that make a pI higher are puton the same “strand” or “monomer” to preserve both functionalities.Similarly, for “skew” variants that come in pairs of a set as more fullyoutlined below, the skilled artisan will consider pI in deciding intowhich strand or monomer one set of the pair will go, such that pIseparation is maximized using the pI of the skews as well.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “host cell” in the context of producing a bispecific antibodyaccording to the invention herein is meant a cell that contains theexogeneous nucleic acids encoding the components of the bispecificantibody and is capable of expressing the bispecific antibody undersuitable conditions. Suitable host cells are discussed below.

By “wild type” or “WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

Provided herein are a number of antibody domains (e.g., Fc domains) thathave sequence identity to human antibody domains. Sequence identitybetween two similar sequences (e.g., antibody variable domains) can bemeasured by algorithms such as that of Smith, T. F. & Waterman, M.S.(1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [localhomology algorithm]; Needleman, S. B. & Wunsch, CD. (1970) “A GeneralMethod Applicable To The Search For Similarities In The Amino AcidSequence Of Two Proteins,” J. Mol. Biol. 48:443 [homology alignmentalgorithm], Pearson, W.R. & Lipman, D. J. (1988) “Improved Tools ForBiological Sequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444[search for similarity method]; or Altschul, S. F. et al, (1990) “BasicLocal Alignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST”algorithm, see https://blast.ncbi.nlm.nih.gov/Blast.cgi. When using anyof the aforementioned algorithms, the default parameters (for Windowlength, gap penalty, etc) are used. In one embodiment, sequence identityis done using the BLAST algorithm, using default parameters

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells, and they can be isolated as well.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacore, SPRor BLI assay.

IV. CD28 and B7H3 Antigen Binding Domains

Provided herein are antigen binding domains (ABDs) and ABD compositionsthat bind either B7H3 or CD28. In some embodiments, one or more of theABDs are included in an antibody format described herein including, forexample, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common lightchain,” and “2+1 common light chain” antibodies.

A. CD28 Antigen Binding Domains and Antibodies

In one aspect, provided herein are CD28 antigen binding domains (ABDs)that bind human CD28, and compositions that include such CD28 antigenbinding domains (e.g., antibodies, including the heterodimericantibodies provided herein). In some embodiments, the CD28 antigenbinding domain described herein are agonistic CD28 ABDs thatadvantageously provide costimulatory activity. Thus, such CD28 ABDsprovided herein are useful of enhancing immune responses, for example,when used as a monotherapy or in combination with other therapeutics(e.g., anti-cancer therapeutics for the treatment of particularcancers).

As will be appreciated by those in the art, suitable CD28 bindingdomains can comprise a set of 6 CDRs as depicted in the Sequence Listingand figures, either as they are underlined or, in the case where adifferent numbering scheme is used as described herein and as shown inTable 2, as the CDRs that are identified using other alignments withinthe variable heavy (VH) domain and variable light domain (VL) sequencesof those depicted in FIGS. 18-21 and 23 and the Sequence Listing.Suitable CD28 ABDs can also include the entire VH and VL sequences asdepicted in these sequences and figures, used as scFvs or as Fabs.

In one embodiment, the CD28 antigen binding domain includes the 6 CDRs(i.e., vhCDR1-3 and vlCDR1-3) of any of the CD28 binding domainsdescribed herein, including the figures and sequence listing. In someembodiments, the CD28 ABD that binds human CD28 is one of the followingCD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23and the Sequence Listing). In exemplary embodiments, the CD28 ABD isCD28 ABDs: 1A7[CD28]_H1L1 or 1A7[CD28]_H1.14L1.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to CD28, provided herein are variantCD28 ABDS having CDRs that include at least one modification of the CD28ABD CDRs disclosed herein (e.g., FIGS. 18-21 and 23 and the SequenceListing). In one embodiment, the CD28 ABD includes a set of 6 CDRs with1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared tothe 6 CDRs of a CD28 ABD as described herein, including the figures andsequence listing. In exemplary embodiments, the CD28 ABD includes a setof 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications ascompared to the 6 CDRs of one of the following CD28 ABDs:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (FIGS. 18-21 and23 and the Sequence Listing). In exemplary embodiments, the CD28 ABD isCD28 ABDs: 1A7[CD28]_H1L1 or 1A7[CD28]_H1.14L1.

In certain embodiments, the CD28 ABD is capable of binding CD28 antigen,as measured by at least one of a Biacore, surface plasmon resonance(SPR) and/or BLI (biolayer interferometry, e.g., Octet assay) assay,with the latter finding particular use in many embodiments. Inparticular embodiments, the CD28 ABD is capable of binding human CD28antigen (see FIG. 1 ).

In some embodiments, the CD28 ABD includes 6 CDRs that are at least 90,95, 97, 98 or 99% identical to the 6 CDRs of a CD28 ABD as describedherein, including the figures and sequence listing. In exemplaryembodiments, the CD28 ABD includes 6 CDRs that are at least 90, 95, 97,98 or 99% identical to the 6 CDRs of one of the following CD28 ABDs:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (FIGS. 18-21 and23 and the Sequence Listing). In certain embodiments, the CD28 ABD iscapable of binding to the CD28, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the CD28ABD is capable of binding human CD28 antigen (see FIG. 1 ).

In another exemplary embodiment, the CD28 ABD include the variable heavy(VH) domain and variable light (VL) domain of any one of the CD28 ABDsdescribed herein, including the figures and sequence listing. Inexemplary embodiments, the CD28 ABD is one of the following CD28 ABDs:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (FIGS. 18-21 and23 and the Sequence Listing).

In addition to the parental CD28 variable heavy and variable lightdomains disclosed herein, provided herein are CD28 ABDs that include avariable heavy domain and/or a variable light domain that are variantsof a CD28 ABD VH and VL domain disclosed herein. In one embodiment, thevariant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acid changes from a VH and/or VL domain of a CD28 ABD describedherein, including the figures and sequence listing. In exemplaryembodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of oneof the following CD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1,1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71,CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1,341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, andhu9.3[CD28]_H1L1 (FIGS. 18-21 and 23 and the Sequence Listing). Incertain embodiments, the CD28 ABD is capable of binding to CD28, asmeasured at least one of a Biacore, surface plasmon resonance (SPR)and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with thelatter finding particular use in many embodiments. In particularembodiments, the CD28 ABD is capable of binding human CD28 antigen (seeFIG. 1 ).

In one embodiment, the variant VH and/or VL domain is at least 90, 95,97, 98 or 99% identical to the VH and/or VL of a CD28 ABD as describedherein, including the figures and sequence listing. In exemplaryembodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98or 99% identical to the VH and/or VL of one of the following CD28 ABDs:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, and hu9.3[CD28]_H1L1 (FIGS. 18-21 and23 and the Sequence Listing). In certain embodiments, the CD28 ABD iscapable of binding to CD28, as measured by at least one of a Biacore,surface plasmon resonance (SPR) and/or BLI (biolayer interferometry,e.g., Octet assay) assay, with the latter finding particular use in manyembodiments. In particular embodiments, the CD28 ABD is capable ofbinding human CD28 antigen (see FIG. 1 ).

In one embodiment, the CD28 antigen binding domain includes a variableheavy domain (V_(H)) having the vhCDR1-3 (i.e., vhCDR1-3) of 1A7_H1.14(FIG. 19 ). In some embodiments, the CD28 antigen binding domain furtherincludes any of the CD28 binding domain variable light domains providedherein. In exemplary embodiments, the variable light domain is 1A7_L1(FIG. 18 ) or a variant thereof. In certain embodiments, the CD28 ABD iscapable of binding CD28 antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the CD28ABD is capable of binding human CD28 antigen (see FIG. 1 ). Such CD28binding domains can be included in any of the antibodies provided hereinincluding, for example, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1common light chain,” and “2+1 common light chain” antibodies.

In one embodiment, the CD28 ABD includes a variable heavy domain (V_(H))having vhCDR1-3s with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidmodifications as compared to the vhCDR1-3 of 1A7_H1.14 (FIG. 19 ). Insome embodiments, the CD28 antigen binding domain further includes anyof the CD28 binding domain variable light domains provided herein. Inexemplary embodiments, the variable light domain is 1A7_L1 (FIG. 18 ) ora variant thereof. In certain embodiments, the CD28 ABD is capable ofbinding CD28 antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octetassay) assay, with the latter finding particular use in manyembodiments. In particular embodiments, the CD28 ABD is capable ofbinding human CD28 antigen (see FIG. 1 ). Such CD28 binding domains canbe included in any of the antibodies provided herein including, forexample, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common lightchain,” and “2+1 common light chain” antibodies.

In some embodiments, the CD28 ABD includes a variable heavy domain(V_(H)) having vhCDR1-3s that are at least 90, 95, 97, 98 or 99%identical to the 6 vhCDR1-3 of 1A7_H1.14 (FIG. 19 ). In someembodiments, the CD28 antigen binding domain further includes any of theCD28 binding domain variable light domains provided herein. In exemplaryembodiments, the variable light domain is 1A7_L1 (FIG. 18 ) or a variantthereof. In certain embodiments, the CD28 ABD is capable of binding tothe CD28, as measured by at least one of a Biacore, surface plasmonresonance (SPR) and/or BLI (biolayer interferometry, e.g., Octet assay)assay, with the latter finding particular use in many embodiments. Inparticular embodiments, the CD28 ABD is capable of binding human CD28antigen (see FIG. 1 ). Such CD28 binding domains can be included in anyof the antibodies provided herein including, for example, “1+1Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1common light chain” antibodies.

In another exemplary embodiment, the CD28 ABD include the variable heavy(V_(H)) domain 1A7_H1.14 (FIG. 19 ). In some embodiments, the CD28antigen binding domain further includes any of the CD28 binding domainvariable light domains provided herein. In exemplary embodiments, thevariable light domain is 1A7_L1 (FIG. 18 ) or a variant thereof. Incertain embodiments, the CD28 ABD is capable of binding to the CD28, asmeasured by at least one of a Biacore, surface plasmon resonance (SPR)and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with thelatter finding particular use in many embodiments. In particularembodiments, the CD28 ABD is capable of binding human CD28 antigen (seeFIG. 1 ). Such CD28 binding domains can be included in any of theantibodies provided herein including, for example, “1+1 Fab-scFv-Fc,”“2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1 common lightchain” antibodies.

In addition to the parental CD28 variable heavy domains disclosedherein, provided herein are CD28 ABDs that include a variable heavydomain that is a variant of 1A7_H1.14 (FIG. 16 ). In one embodiment, thevariant VH domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidchanges from 1A7_H1.14 (FIG. 19 ). In some embodiments, the CD28 antigenbinding domain further includes any of the CD28 binding domain variablelight domains provided herein. In exemplary embodiments, the variablelight domain is 1A7_L1 (FIG. 18 ) or a variant thereof. In certainembodiments, the CD28 ABD is capable of binding to CD28, as measured atleast one of a Biacore, surface plasmon resonance (SPR) and/or BLI(biolayer interferometry, e.g., Octet assay) assay, with the latterfinding particular use in many embodiments. In particular embodiments,the CD28 ABD is capable of binding human CD28 antigen (see FIG. 1 ).Such CD28 binding domains can be included in any of the antibodiesprovided herein including, for example, “1+1 Fab-scFv-Fc,” “2+1Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1 common light chain”antibodies.

In one embodiment, the variant VH domain is at least 90, 95, 97, 98 or99% identical to 1A7_H1.14 (FIG. 19 ). In some embodiments, the CD28antigen binding domain further includes any of the CD28 binding domainvariable light domains provided herein. In exemplary embodiments, thevariable light domain is 1A7_L1 (FIG. 18 ) or a variant thereof. Incertain embodiments, the CD28 ABD is capable of binding to CD28, asmeasured by at least one of a Biacore, surface plasmon resonance (SPR)and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with thelatter finding particular use in many embodiments. In particularembodiments, the CD28 ABD is capable of binding human CD28 antigen (seeFIG. 1 ). Such CD28 binding domains can be included in any of theantibodies provided herein including, for example, “1+1 Fab-scFv-Fc,”“2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1 common lightchain” antibodies.

Specific anti-CD28 ABDs of interest include a VH domain with an aminoacid sequence selected from the group consisting of SEQ ID NO:870, SEQID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589,SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ IDNO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603,SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ IDNO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617,SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ IDNO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:625, SEQ ID NO:626, SEQID NO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631,SEQ ID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ IDNO:636, SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQID NO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645,SEQ ID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ IDNO:650, SEQ ID NO:651, SEQ ID NO: 1198 and SEQ ID NO: 1199, paired witha VL domain of SEQ ID NO:874.

In other cases, the anti-CD28 VH domain has an amino acid sequenceselected from SEQ ID NO:870, SEQ ID NO:585, SEQ ID NO:586, SEQ IDNO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596,SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ IDNO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610,SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ IDNO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624,SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ IDNO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638,SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ IDNO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:1198and SEQ ID NO:1199, and a VL domain with an amino acid sequence selectedfrom the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQ IDNO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662,SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ IDNO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676,SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ IDNO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690,SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ IDNO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704,SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ IDNO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718,SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ IDNO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732,SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ IDNO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746,SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ IDNO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQID NO:1200 and SEQ ID NO:756.

In some cases, the anti-CD28 binding domain has a VH domain and VLdomain with amino acid sequences selected from the pairs of a) SEQ IDNOs:1 and 5, b) SEQ ID NOs: 9 and 13, c) SEQ ID NOs:17 and 21, d) SEQ IDNOs:25 and 29, e) SEQ ID NOs:33 and 37, f) SEQ ID NOs:41 and 45; g) SEQID NOs:49 and 53, h) SEQ ID NOs:57 and 61, i) SEQ ID NOs:65 and 69, j)SEQ ID NOs:73 and 77, and k) SEQ ID NOs:81 and 85.

B. B7H3 Antigen Binding Domains

In one aspect, provided herein are B7H3 antigen binding domains (ABDs)and compositions that include such B7H3 antigen binding domains (ABDs),including anti-B7H3 antibodies. Such B7H3 binding domains and relatedantibodies (e.g., anti-B7H3×anti-CD28 bispecific antibodies) find use,for example, in the treatment of B7H3 associated cancers.

As will be appreciated by those in the art, suitable B7H3 bindingdomains can comprise a set of 6 CDRs as depicted in the Sequence Listingand FIGS. 26-31 , either as the CDRs are underlined or, in the casewhere a different numbering scheme is used as described herein and asshown in Table 2, as the CDRs that are identified using other alignmentswithin the variable heavy (V_(H)) domain and variable light domain(V_(L)) sequences of those depicted in FIGS. 26-31 and the SequenceListing (see Table 2). Suitable B7H3 ABDs can also include the entire VHand VL sequences as depicted in these sequences and figures, used asscFvs or as Fab domains.

In one embodiment, the B7H3 antigen binding domain includes the 6 CDRs(i.e., vhCDR1-3 and vlCDR1-3) of a B7H3 ABD described herein, includingthe figures and sequence listing. In exemplary embodiments, the B7H3 ABDis one of the following B7H3 ABDs: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704 (FIGS. 26-31 and theSequence Listing).

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to B7H3, provided herein are variantB7H3 ABDS having CDRs that include at least one modification of the B7H3ABD CDRs disclosed herein. In one embodiment, the B7H3 ABD includes aset of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidmodifications as compared to the 6 CDRs of a B7H3 ABD described herein,including the figures and sequence listing. In exemplary embodiments,the B7H3 ABD includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10amino acid modifications as compared to the 6 CDRs of one of thefollowing B7H3 ABDs: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704 (FIGS. 26-31 and theSequence Listing). In certain embodiments, the variant B7H3 ABD iscapable of binding B7H3 antigen, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the B7H3ABD is capable of binding human B7H3 antigen (see FIG. 2 ).

In one embodiment, the B7H3 ABD includes 6 CDRs that are at least 90,95, 97, 98 or 99% identical to the 6 CDRs of a B7H3 ABD as describedherein, including the figures and sequence listing. In exemplaryembodiments, the B7H3 ABD includes 6 CDRs that are at least 90, 95, 97,98 or 99% identical to the 6 CDRs of one of the following B7H3 ABDs:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704 (FIGS. 26-31 and the Sequence Listing). In certain embodiments,the B7H3 ABD is capable of binding to B7H3 antigen, as measured by atleast one of a Biacore, surface plasmon resonance (SPR) and/or BLI(biolayer interferometry, e.g., Octet assay) assay, with the latterfinding particular use in many embodiments. In particular embodiments,the B7H3 ABD is capable of binding human B7H3 antigen (see FIG. 2 ).

In another exemplary embodiment, the B7H3 ABD include the variable heavy(V_(H)) domain and variable light (V_(L)) domain of any one of the B7H3ABDs described herein, including the figures and sequence listing. Inexemplary embodiments, the B7H3 ABD is one of the following B7H3 ABDs:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704 (FIGS. 26-31 and theSequence Listing). In exemplary embodiments, the B7H3 ABD is one of thefollowing: B7H3 ABDs: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, or6A1[B7H3]_H1L1.

In addition to the parental B7H3 variable heavy and variable lightdomains disclosed herein, provided herein are B7H3 ABDs that include avariable heavy domain and/or a variable light domain that are variantsof a B7H3 ABD VH and VL domain disclosed herein. In one embodiment, thevariant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acid changes from a VH and/or VL domain of a B7H3 ABD describedherein, including the figures and sequence listing. In exemplaryembodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of oneof the following B7H3 ABDs: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704 (FIGS. 26-31 and theSequence Listing). In certain embodiments, the B7H3 ABD is capable ofbinding to B7H3, as measured at least one of a Biacore, surface plasmonresonance (SPR) and/or BLI (biolayer interferometry, e.g., Octet assay)assay, with the latter finding particular use in many embodiments. Inparticular embodiments, the B7H3 ABD is capable of binding human B7H3antigen (see FIG. 2 ).

In one embodiment, the variant VH and/or VL domain is at least 90, 95,97, 98 or 99% identical to the VH and/or VL of a B7H3 ABD as describedherein, including the figures and sequence listing. In exemplaryembodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98or 99% identical to the VH and/or VL of one of the following B7H3 ABDs:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704 (FIGS. 26-31 and the Sequence Listing). In certain embodiments,the B7H3 ABD is capable of binding to the B7H3, as measured by at leastone of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the B7H3ABD is capable of binding human B7H3 antigen (see FIG. 2 ).

In one embodiment, the B7H3 antigen binding domain includes a variableheavy domain (V_(H)) having the vhCDR1-3 (i.e., vhCDR1-3) of2E4A3.189_H1.22 (FIG. 27 ). In some embodiments, the B7H3 antigenbinding domain further includes any of the B7H3 or CD28 binding domainvariable light domains provided herein. In exemplary embodiments, thevariable light domain is 2E4A3.189_L1 (FIG. 26 ), 1A7_L1 (FIG. 18 ) or avariant thereof. In certain embodiments, the B7H3 ABD is capable ofbinding B7H3 antigen, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octetassay) assay, with the latter finding particular use in manyembodiments. In particular embodiments, the B7H3 ABD is capable ofbinding human B7H3 antigen (see FIG. 2 ). Such B7H3 binding domains canbe included in any of the antibodies provided herein including, forexample, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common lightchain,” and “2+1 common light chain” antibodies.

In one embodiment, the B7H3 ABD includes a variable heavy domain (V_(H))having vhCDR1-3s with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidmodifications as compared to the vhCDR1-3 of 2E4A3.189_H1.22 (FIG. 27 ).In some embodiments, the B7H3 antigen binding domain further includesany of the B7H3 or CD28 binding domain variable light domains providedherein. In exemplary embodiments, the variable light domain is2E4A3.189_L1 (FIG. 26 ), 1A7_L1 (FIG. 18 ) or a variant thereof. Incertain embodiments, the B7H3 ABD is capable of binding B7H3 antigen, asmeasured by at least one of a Biacore, surface plasmon resonance (SPR)and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with thelatter finding particular use in many embodiments. In particularembodiments, the B7H3 ABD is capable of binding human B7H3 antigen (seeFIG. 2 ). Such B7H3 binding domains can be included in any of theantibodies provided herein including, for example, “1+1 Fab-scFv-Fc,”“2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1 common lightchain” antibodies.

In some embodiments, the B7H3 ABD includes a variable heavy domain(V_(H)) having vhCDR1-3s that are at least 90, 95, 97, 98 or 99%identical to the 6 vhCDR1-3 of 2E4A3.189_H1.22 (FIG. 27 ). In someembodiments, the B7H3 antigen binding domain further includes any of theB7H3 or CD28 binding domain variable light domains provided herein. Inexemplary embodiments, the variable light domain is 2E4A3.189 L1 (FIG.26 ), 1A7_L1 (FIG. 18 ) or a variant thereof. In certain embodiments,the B7H3 ABD is capable of binding to the B7H3, as measured by at leastone of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the B7H3ABD is capable of binding human B7H3 antigen (see FIG. 2 ). Such B7H3binding domains can be included in any of the antibodies provided hereinincluding, for example, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1common light chain,” and “2+1 common light chain” antibodies.

In another exemplary embodiment, the B7H3 ABD include the variable heavy(V_(H)) domain 2E4A3.189_H1.22 (FIG. 27 ). In some embodiments, the B7H3antigen binding domain further includes any of the B7H3 or CD28 bindingdomain variable light domains provided herein. In exemplary embodiments,the variable light domain is 2E4A3.189_L1 (FIG. 26 ), 1A7_L1 (FIG. 18 )or a variant thereof. In certain embodiments, the B7H3 ABD is capable ofbinding to the B7H3, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octetassay) assay, with the latter finding particular use in manyembodiments. In particular embodiments, the B7H3 ABD is capable ofbinding human B7H3 antigen (see FIG. 1 ). Such B7H3 binding domains canbe included in any of the antibodies provided herein including, forexample, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common lightchain,” and “2+1 common light chain” antibodies.

In addition to the parental B7H3 variable heavy domains disclosedherein, provided herein are B7H3 ABDs that include a variable heavydomain that is a variant of the variable heavy (V_(H)) domain2E4A3.189_H1.22 (FIG. 27 ). In one embodiment, the variant VH domain hasfrom 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from thevariable heavy (V_(H)) domain 2E4A3.189_H1.22 (FIG. 27 ). In someembodiments, the B7H3 antigen binding domain further includes any of theB7H3 or CD28 binding domain variable light domains provided herein. Inexemplary embodiments, the variable light domain is 2E4A3.189_L1 (FIG.26 ), 1A7_L1 (FIG. 18 ) or a variant thereof. In certain embodiments,the B7H3 ABD is capable of binding to B7H3, as measured at least one ofa Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the B7H3ABD is capable of binding human B7H3 antigen (see FIG. 2 ). Such B7H3binding domains can be included in any of the antibodies provided hereinincluding, for example, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1common light chain,” and “2+1 common light chain” antibodies.

In one embodiment, the variant VH domain is at least 90, 95, 97, 98 or99% identical to 2E4A3.189_H1.22 (FIG. 27 ). In some embodiments, theB7H3 antigen binding domain further includes any of the B7H3 or CD28binding domain variable light domains provided herein. In exemplaryembodiments, the variable light domain is 2E4A3.189_L1 (FIG. 26 ),1A7_L1 (FIG. 18 ) or a variant thereof. In certain embodiments, the B7H3ABD is capable of binding to B7H3, as measured by at least one of aBiacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments. In particular embodiments, the B7H3ABD is capable of binding human B7H3 antigen (see FIG. 2 ). Such B7H3binding domains can be included in any of the antibodies provided hereinincluding, for example, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1common light chain,” and “2+1 common light chain” antibodies.

In some embodiments, the anti-B7H3 ABD has a VH domain and VL domainwith amino acid sequences selected from the pairs of a) SEQ ID NOs: 89and 93 from omburamab, b) SEQ ID NOs:97 and 101 from enoblituzumab, c)SEQ ID NOs:105 and 109 from BRCA84D, d) SEQ ID NOs:113 and 117 fromBRCA69D, e) SEQ ID NOs:121 and 125 from PRCA157, f) SEQ ID NOs:129 and133 from huPRCA157, g) SEQ ID NOs:137 and 141 from Mab-D; h) SEQ IDNOs:145 and 149 humAb-D; i) SEQ ID NOs:153 and 157 from m30; j) SEQ IDNOs:161 and 165 from M30-H1-L4, k) SEQ ID NOs:169 and 173 SP265; l) SEQID NOs:177 and 181 from S10-H50L58; m) SEQ ID NOs:185 and 189 from 8H9,n) SEQ ID NOs:193 and 197 from m852; o) SEQ ID NOs:201 and 205 fromm857; p) SEQ ID NOs:209 and 213 from m8524; q) SEQ ID NOs:217 and 221from 1-1; r) SEQ ID NOs:225 and 229 from 1-2; s) SEQ ID NOs:233 and 237from 1-4; t) SEQ ID NOs:241 and 245 from 1-5; u) SEQ ID NOs:249 and 253from 1-7; v) SEQ ID NOs:257 and 261 from 2-5; w) SEQ ID NOs:265 and 269from 2-8; x) SEQ ID NOs: 273 and 277 from chAb2; y) SEQ ID NOs:281 and285 chAb3; z) SEQ ID NOs:289 and 293 from chAb4; aa) SEQ ID NOs:297 and301 from chAb18; bb) SEQ ID NOs:305 and 309 from chAb13; cc) SEQ IDNOs:313 and 317 from chAb12; dd) SEQ ID NOs:321 and 325 from chAb14; ee)SEQ ID NOs:329 and 333 from chAb6; ff) SEQ ID NOs:337 and 341 fromchAb11, gg) SEQ ID NOs:345 and 349 from chAB16; hh) SEQ ID NOs:353 and357 from chAb10; ii) SEQ ID NOs:361 and 365 from ChAb7; jj) SEQ IDNOs:369 and 373 from chAb8, kk) SEQ ID NOs:377 and 381 from chAb17; ll)SEQ ID NOs:385 and 389 from chAb5, mm) SEQ ID NOs:393 and 397 fromhuAb3v2.5, nn) SEQ ID NOs:401 and 405 from huAb3v2.6, pp) SEQ ID NOs:409and 413 from huAb13v1, qq) SEQ ID NOs:417 and 421 from TPP-5706, rr) SEQID NOs:425 and 429 from TPP-6642; ss) SEQ ID NOs:433 and 437 fromTPP-6850, tt) SEQ ID NOs:441 and 445 from TPP-3803, uu) SEQ ID NOs:449and 453 from TRL4542, vv) SEQ ID NOs:457 and 461 from h1702, ww) SEQ IDNOs:465 and 469 from h1703, xx) SEQ ID NOs:473 and 477 from huA3, yy)SEQ ID NOs:481 and 485 from huA9 and zz) SEQ ID NOs: 489 and 493 fromm1704. See FIG. 17 from U.S. Ser. No. 63/092,272.

In some embodiments, the anti-B7H3 ABD has an VH domain with the aminoacid sequence of SEQ ID NO:942 (2E4A3.189_H1.22) and a VL domain withthe amino acid sequence of SEQ ID NO:874 (1A7[CD28]_L1, which is thecommon light chain for both B7H3 and CD28).

V. Antibodies

In one aspect provided herein are anti-CD28 antibodies and anti-B7H3antibodies. Antibodies provided herein can include any of the B7H3and/or CD28 binding domains provided herein (e.g., “1+1 Fab-scFv-Fc,”“2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and “2+1 common lightchain” antibodies).

The antibodies provided herein include different antibody domains. Asdescribed herein and known in the art, the antibodies described hereininclude different domains within the heavy and light chains, which canbe overlapping as well. These domains include, but are not limited to,the Fc domain, the CH1 domain, the CH2 domain, the CH3 domain, the hingedomain, the heavy constant domain (CH1-hinge-Fc domain orCH1-hinge-CH2-CH3), the variable heavy domain, the variable lightdomain, the light constant domain, Fab domains and scFv domains.

As shown herein, there are a number of suitable linkers (for use aseither domain linkers or scFv linkers) that can be used to covalentlyattach the recited domains (e.g., scFvs, Fabs, Fc domains, etc.),including traditional peptide bonds, generated by recombinanttechniques. Exemplary linkers to attach domains of the subject antibodyto each other are depicted in FIG. 7 . In some embodiments, the linkerpeptide may predominantly include the following amino acid residues:Gly, Ser, Ala, or Thr. The linker peptide should have a length that isadequate to link two molecules in such a way that they assume thecorrect conformation relative to one another so that they retain thedesired activity. In one embodiment, the linker is from about 1 to 50amino acids in length, preferably about 1 to 30 amino acids in length.In one embodiment, linkers of 1 to 20 amino acids in length may be used,with from about 5 to about 10 amino acids finding use in someembodiments. Useful linkers include glycine-serine polymers, includingfor example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is aninteger of at least one (and generally from 3 to 4), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers.Alternatively, a variety of nonproteinaceous polymers, including but notlimited to polyethylene glycol (PEG), polypropylene glycol,polyoxyalkylenes, or copolymers of polyethylene glycol and polypropyleneglycol, may find use as linkers.

Other linker sequences may include any sequence of any length of C/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the C/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cx or Ck. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g., TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains as outlined herein together. For example, in the 2+1Fab₂-scFv-Fc format, there may be a domain linker that attaches theC-terminus of the CH1 domain of the Fab to the N-terminus of the scFv,with another optional domain linker attaching the C-terminus of the scFvto the CH2 domain (although in many embodiments the hinge is used asthis domain linker). While any suitable linker can be used, manyembodiments utilize a glycine-serine polymer as the domain linker,including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n isan integer of at least one (and generally from 3 to 4 to 5) as well asany peptide sequence that allows for recombinant attachment of the twodomains with sufficient length and flexibility to allow each domain toretain its biological function. In some cases, and with attention beingpaid to “strandedness”, as outlined below, charged domain linkers, asused in some embodiments of scFv linkers can be used. Exemplary usefuldomain linkers are depicted in FIG. 7 .

In some embodiments, the linker is a scFv linker that is used tocovalently attach the VH and VL domains as discussed herein. In manycases, the scFv linker is a charged scFv linker, a number of which areshown in FIG. 6 . Accordingly, provided herein are charged scFv linkers,to facilitate the separation in pI between a first and a second monomer.That is, by incorporating a charged scFv linker, either positive ornegative (or both, in the case of scaffolds that use scFvs on differentmonomers), this allows the monomer comprising the charged linker toalter the pI without making further changes in the Fc domains. Thesecharged linkers can be substituted into any scFv containing standardlinkers. Again, as will be appreciated by those in the art, charged scFvlinkers are used on the correct “strand” or monomer, according to thedesired changes in pI. For example, as discussed herein, to make 1+1Fab-scFv-Fc format heterodimeric antibody, the original pI of the Fvregion for each of the desired antigen binding domains are calculated,and one is chosen to make an scFv, and depending on the pI, eitherpositive or negative linkers are chosen.

Charged domain linkers can also be used to increase the pI separation ofthe monomers of the invention as well, and thus those included in FIG. 6can be used in any embodiment herein where a linker is utilized.

The B7H3 binding domains and CD28 binding domains provided can beincluded in any useful antibody format including, for example, canonicalimmunoglobulin, as well as the “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,”“1+1 common light chain,” and “2+1 common light chain” formats providedherein (see, e.g., FIG. 25 ). Other useful antibody formats include, butare not limited to, “mAb-Fv,” “mAb-scFv,” “central-Fv”, “one armedscFv-mAb,” “scFv-mAb,” “dual scFv,” and “trident” format antibodies, asdisclosed in US20180127501A1, which is incorporated by reference herein,particularly in pertinent part relating to antibody formats (see, e.g.,FIG. 2 ).

In some embodiments, the subject antibody includes one or more of theB7H3 ABDs provided herein. In some embodiments, the antibody includesone B7H3 ABD. In other embodiments, the antibody includes two B7H3 ABDs.In exemplary embodiments, the B7H3 ABD includes the variable heavydomain and variable light domain of one of the following B7H3 ABDs:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, andm1704 (FIGS. 26-31 and the Sequence Listing). In some embodiments, theB7H3 ABD is one of the following B7H3 ABDs: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, m1704 (FIGS. 26-31 and the SequenceListing).

In an exemplary embodiment, the antibody is a bispecific antibody thatincludes one or two B7H3 ABDs, including any of the B7H3 ABDs providedherein. Bispecific antibody that include such B7H3 ABDs include, forexample, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common lightchain,” and “2+1 common light chain” bispecifics format antibodies (FIG.25 ). In exemplary embodiments, the B7H3 ABD is one of the followingB7H3 ABDs: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704(FIGS. 26-31 and the Sequence Listing). In exemplary embodiments theB7H3 binding domains is a Fab. In some embodiments, such bispecificantibodies are heterodimeric bispecific antibodies that include any ofthe heterodimerization skew variants, pI variants and/or ablationvariants described herein. See FIG. 8 .

In some embodiments, the subject antibody includes one or more of theCD28 ABDs provided herein. In some embodiments, the antibody includesone CD28 ABD. In other embodiments, the antibody includes two CD28 ABDs.In exemplary embodiments, the antibody includes the variable heavydomain and variable light domain of one of the CD28 ABDs:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23and the Sequence Listing).

In an exemplary embodiment, the antibody is a bispecific antibody thatincludes one or two CD28 ABDs, including any of the CD28 ABDs providedherein. Bispecific antibody that include such CD28 ABDs include, forexample, “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common lightchain,” and “2+1 common light chain” bispecifics format antibodies (FIG.25 ). In exemplary embodiments, the CD28 ABD is one of the followingCD28 ABDs: 1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,hCD28.3[CD28]_H1L1, 5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1,341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1,PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23and the Sequence Listing). In exemplary embodiments, the CD28 ABD is ananti-CD28 scFv included in an “1+1 Fab-scFv-Fc,” or “2+1 Fab₂-scFv-Fcbispecifics format antibodies (FIG. 25 ). In some embodiments, suchbispecific antibodies are heterodimeric bispecific antibodies thatinclude any of the heterodimerization skew variants, pI variants and/orablation variants described herein. See FIG. 8 .

A. Chimeric and Humanized Antibodies

In certain embodiments, the subject antibodies provided herein include aheavy chain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchantibodies may comprise or consist of a human antibody comprising heavyor light chain variable regions that are “the product of” or “derivedfrom” a particular germline sequence. A human antibody that is “theproduct of” or “derived from” a human germline immunoglobulin sequencecan be identified as such by comparing the amino acid sequence of thehuman antibody to the amino acid sequences of human germlineimmunoglobulins and selecting the human germline immunoglobulin sequencethat is closest in sequence (i.e., greatest % identity) to the sequenceof the human antibody (using the methods outlined herein). A humanantibody that is “the product of” or “derived from” a particular humangermline immunoglobulin sequence may contain amino acid differences ascompared to the germline sequence, due to, for example,naturally-occurring somatic mutations or intentional introduction ofsite-directed mutation. However, a humanized antibody typically is atleast 90% identical in amino acids sequence to an amino acid sequenceencoded by a human germline immunoglobulin gene and contains amino acidresidues that identify the antibody as being derived from humansequences when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a humanized antibody may be at least 95, 96, 97, 98 or 99%, oreven at least 96%, 97%, 98%, or 99% identical in amino acid sequence tothe amino acid sequence encoded by the germline immunoglobulin gene.Typically, a humanized antibody derived from a particular human germlinesequence will display no more than 10-20 amino acid differences from theamino acid sequence encoded by the human germline immunoglobulin gene(prior to the introduction of any skew, pI and ablation variants herein;that is, the number of variants is generally low, prior to theintroduction of the variants of the invention). In certain cases, thehumanized antibody may display no more than 5, or even no more than 4,3, 2, or 1 amino acid difference from the amino acid sequence encoded bythe germline immunoglobulin gene (again, prior to the introduction ofany skew, pI and ablation variants herein; that is, the number ofvariants is generally low, prior to the introduction of the variants ofthe invention).

In one embodiment, the parent antibody has been affinity matured, as isknown in the art. Structure-based methods may be employed forhumanization and affinity maturation, for example as described in U.S.Ser. No. 11/004,590. Selection based methods may be employed to humanizeand/or affinity mature antibody variable regions, including but notlimited to methods described in Wu et al., 1999, J. Mol. Biol.294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al.,1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003,Protein Engineering 16(10):753-759, all entirely incorporated byreference. Other humanization methods may involve the grafting of onlyparts of the CDRs, including but not limited to methods described inU.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125;De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirelyincorporated by reference.

B. Anti-CD28×Anti-Tumor Associated Antigen (TAA) Antibodies

In another aspect, provided herein are anti-CD28×anti-TAA antibodies. Insome embodiments, the anti-CD28×anti-TAA antibody includes a CD28binding and one or more binding domains that bind a tumor associatedantigen. In some embodiments, the CD28 binding domain of the antibody isan agonistic CD28 binding domain that provides co-stimulatory functionby binding to CD28 on T cells. As such, the anti-CD28×anti-TAA antibodyprovided herein enhance immune responses selectively at tumor sites thatexpress the particular TAA (e.g., B7H3). In some embodiments, theanti-CD28×anti-TAA antibody is a bispecific antibody. In someembodiments, the anti-CD28×anti-TAA antibody is a trispecific antibody.In some embodiments, the anti-CD28×anti-TAA antibody is a bivalentantibody. In some embodiments, the anti-CD28×anti-TAA antibody is atrivalent antibody. In some embodiments, the anti-CD28×anti-TAA antibodyis a bispecific, bivalent antibody. In exemplary embodiments, theanti-CD28×anti-TAA antibody is a bispecific, trivalent antibody.

As is more fully outlined herein, the anti-CD28×anti-TAA antibody can bein a variety of formats, as outlined below. Exemplary formats includethe “1+1 Fab-scFv-Fc,” “2+1 Fab₂-scFv-Fc,” “1+1 common light chain,” and“2+1 common light chain” formats provided herein (see, e.g., FIG. 25 ).Other useful antibody formats include, but are not limited to, “mAb-Fv,”“mAb-scFv,” “central-Fv”, “one armed scFv-mAb,” “scFv-mAb,” “dual scFv,”and “trident” format antibodies, as disclosed in US20180127501A1, whichis incorporated by reference herein, particularly in pertinent partrelating to antibody formats (see, e.g., FIG. 2 ).

The anti-CD28×anti-TAA antibody can include any suitable CD28 ABD,including those described herein. In some embodiments, the CD28 ABD isan agonistic ABD that provides co-stimulatory function upon binding toCD28. In some embodiments, the anti-CD28×anti-TAA antibody includes aCD28 binding domain that includes the variable heavy domain and variablelight of one of the following CD28 binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,and hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23 and Sequence Listing) orvariant thereof.

The anti-CD28×anti-TAA antibody provided herein can include one or moreTAA binding domains. In some embodiments, the anti-CD28×anti-TAAantibody includes one TAA binding domain. In certain embodiments, theanti-CD28×anti-TAA antibody includes two TAA binding domain. Anysuitable TAA binding domain can be included in the subjectanti-CD28×anti-TAA antibody, depending on the tumor selected fortargeting. TAAs that can be targeted by the anti-CD28×anti-TAAantibodies provided herein include, but are not limited to: B7H, CD20,CD38, CD123; ROR1, ROR2, BCMA; PSMA; SSTR2; SSTR5, CD19, FLT3, CD33,PSCA, ADAM 17, CEA, Her2, EGFR, EGFR-vIII, CD30, FOLR1, GD-2, CA-IX,Trop-2, CD70, CD38, mesothelin, EphA2, CD22, CD79b, GPNMB, CD56, CD138,CD52, CD74, CD30, CD123, RON, ERBB2, and EGFR. Additional TAAs aredescribed for example, in US20160355608 and US20170209492, which areincorporated herein in pertinent parts relating to tumor-associatedantigens. Suitable TAA binding domains that can be included in thesubject anti-CD28×anti-TAA antibodies are disclosed, for example,US20190248898A1 (SSTR2), US20200165356A1 (FAP), US20170320947A1 (PSMA),which are all incorporated by reference in pertinent parts relating toTAA binding domains.

In certain embodiments, the anti-CD28×anti-TAA antibody includes a B7H3binding domain. In some embodiments, such anti-CD28×anti-B7H3 (alsoreferred to herein as “αB7H3ΔαCD28” or as “αCD28×αB7H3”) bispecificantibodies include at least one B7H3 ABD and at least one CD28 bindingdomain. In exemplary embodiments, the anti-CD28×anti-B7H3 bispecificantibody includes two B7H3 binding domains. In some embodiments, theCD28 binding domain of the bispecific antibody is an agonistic CD28binding domain that provides co-stimulatory function by binding to CD28on T cells. As such, the bispecific αB7H3ΔαCD28 provided herein enhanceimmune responses selectively in tumor sites that express B7H3.

The anti-CD28×anti-B7H3 bispecific antibody can include any suitableCD28 ABD and B7H3 ABD, including those described herein. In someembodiments, the anti-CD28×anti-B7H3 bispecific antibody includes a CD28binding domain that includes the variable heavy domain and variablelight of one of the following CD28 binding domains: 1A7[CD28]_H1L1,1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, hCD28.3[CD28]_H1L1,5.11A1[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23 and Sequence Listing) or variantthereof. In some embodiments, the B7H3 ABD includes the variable heavydomain and variable light domain of one of the following B7H3 ABDs:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704 (FIGS. 26-31 and theSequence Listing) or variants thereof.

Note that unless specified herein, the order of the antigen list in thename does not confer structure; that is an anti-B7H3 X anti-CD28 1+1Fab-scFv-Fc antibody can have the scFv bind to B7H3 or CD28, although insome cases, the order specifies structure as indicated.

In addition, in embodiments wherein the subject antibody includes anscFv, the scFv can be in an orientation from N- to C-terminus ofV_(H)-scFv linker-VL or V_(L)-scFv linker-V_(H). In some formats, one ormore of the ABDs generally is a Fab that includes a VH domain on oneprotein chain (generally as a component of a heavy chain) and a VL onanother protein chain (generally as a component of a light chain).

As will be appreciated by those in the art, any set of 6 CDRs or VH andVL domains can be in the scFv format or in the Fab format, which is thenadded to the heavy and light constant domains, where the heavy constantdomains comprise variants (including within the CH1 domain as well asthe Fc domain). The scFv sequences contained in the sequence listingutilize a particular charged linker, but as outlined herein, unchargedor other charged linkers can be used, including those depicted in FIG. 6.

In addition, as discussed above, the numbering used in the SequenceListing for the identification of the CDRs is Kabat, however, differentnumbering can be used, which will change the amino acid sequences of theCDRs as shown in Table 2.

For all of the variable heavy and light domains listed herein, furthervariants can be made. As outlined herein, in some embodiments the set of6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (withamino acid substitutions finding particular use), as well as changes inthe framework regions of the variable heavy and light domains, as longas the frameworks (excluding the CDRs) retain at least about 80, 85 or90% identity to a human germline sequence selected from those listed inFIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend isincorporated by reference in its entirety herein. Thus, for example, theidentical CDRs as described herein can be combined with differentframework sequences from human germline sequences, as long as theframework regions retain at least 80, 85 or 90% identity to a humangermline sequence selected from those listed in FIG. 1 of U.S. Pat. No.7,657,380. Alternatively, the CDRs can have amino acid modifications(e.g., from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs(that is, the CDRs can be modified as long as the total number ofchanges in the set of 6 CDRs is less than 6 amino acid modifications,with any combination of CDRs being changed; e.g., there may be onechange in vlCDR1, two in vhCDR2, none in vhCDR3, etc.)), as well ashaving framework region changes, as long as the framework regions retainat least 80, 85 or 90% identity to a human germline sequence selectedfrom those listed in FIG. 1 of U.S. Pat. No. 7,657,380.

C. Heterodimeric Antibodies

In exemplary embodiments, the anti-CD28×anti-TAA (e.g.,anti-CD28×anti-B7H3) antibodies provided herein are heterodimericbispecific antibodies that include two variant Fc domain sequences. Suchvariant Fc domains include amino acid modifications to facilitate theself-assembly and/or purification of the heterodimeric antibodies.

An ongoing problem in antibody technologies is the desire for“bispecific” antibodies that bind to two different antigenssimultaneously, in general thus allowing the different antigens to bebrought into proximity and resulting in new functionalities and newtherapies. In general, these antibodies are made by including genes foreach heavy and light chain into the host cells. This generally resultsin the formation of the desired heterodimer (A-B), as well as the twohomodimers (A-A and B-B (not including the light chain heterodimericissues)). However, a major obstacle in the formation of bispecificantibodies is the difficulty in biasing the formation of the desiredheterodimeric antibody over the formation of the homodimers and/orpurifying the heterodimeric antibody away from the homodimers.

There are a number of mechanisms that can be used to generate thesubject heterodimeric antibodies. In addition, as will be appreciated bythose in the art, these different mechanisms can be combined to ensurehigh heterodimerization. Amino acid modifications that facilitate theproduction and purification of heterodimers are collectively referred togenerally as “heterodimerization variants.” As discussed below,heterodimerization variants include “skew” variants (e.g., the “knobsand holes” and the “charge pairs” variants described below) as well as“pI variants,” which allow purification of heterodimers from homodimers.As is generally described in U.S. Pat. No. 9,605,084, herebyincorporated by reference in its entirety and specifically as below forthe discussion of heterodimerization variants, useful mechanisms forheterodimerization include “knobs and holes” (“KIH”) as described inU.S. Pat. No. 9,605,084, “electrostatic steering” or “charge pairs” asdescribed in U.S. Pat. No. 9,605,084, pI variants as described in U.S.Pat. No. 9,605,084, and general additional Fc variants as outlined inU.S. Pat. No. 9,605,084 and below.

Heterodimerization variants that are useful for the formation andpurification of the subject heterodimeric antibody (e.g., bispecificantibodies) are further discussed in detailed below.

1. Skew Variants

In some embodiments, the heterodimeric antibody includes skew variantswhich are one or more amino acid modifications in a first Fc domain (A)and/or a second Fc domain (B) that favor the formation of Fcheterodimers (Fc dimers that include the first and the second Fc domain;(A-B) over Fc homodimers (Fc dimers that include two of the first Fcdomain or two of the second Fc domain; A-A or B-B). Suitable skewvariants are included in the FIG. 29 of US Publ. App. No. 2016/0355608,hereby incorporated by reference in its entirety and specifically forits disclosure of skew variants, as well as in FIGS. 3 and 9 .

One particular type of skew variants is generally referred to in the artas “knobs and holes,” referring to amino acid engineering that createssteric influences to favor heterodimeric formation and disfavorhomodimeric formation, as described in U.S. Ser. No. 61/596,846, Ridgwayet al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol.Biol. 1997 270:26; U.S. Pat. No. 8,216,805, all of which are herebyincorporated by reference in their entirety and specifically for thedisclosure of “knobs and holes” mutations. This is sometime referred toherein as “steric variants.” The figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and holes” mutations can be combined with disulfide bonds tofurther favor formation of Fc heterodimers.

Another method that finds use in the generation of heterodimers issometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “skewvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g., these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

In some embodiments, the skew variants advantageously and simultaneouslyfavor heterodimerization based on both the “knobs and holes” mechanismas well as the “electrostatic steering” mechanism. In some embodiments,the heterodimeric antibody includes one or more sets of suchheterodimerization skew variants. These variants come in “pairs” of“sets”. That is, one set of the pair is incorporated into the firstmonomer and the other set of the pair is incorporated into the secondmonomer. It should be noted that these sets do not necessarily behave as“knobs in holes” variants, with a one-to-one correspondence between aresidue on one monomer and a residue on the other. That is, these pairsof sets may instead form an interface between the two monomers thatencourages heterodimer formation and discourages homodimer formation,allowing the percentage of heterodimers that spontaneously form underbiological conditions to be over 90%, rather than the expected 50% (25%homodimer A/A:50% heterodimer A/B:25% homodimer B/B). Exemplaryheterodimerization “skew” variants are depicted in FIG. 4 . In exemplaryembodiments, the heterodimeric antibody includes aS364K/E357Q:L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K;T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L; K370S: S364K/E357Q;or a T366S/L368A/Y407V: T366W (optionally including a bridgingdisulfide, T366S/L368A/Y407V/Y349C: T366W/S354C) “skew” variant aminoacid substitution set. In an exemplary embodiment, the heterodimericantibody includes a “S364K/E357Q:L368D/K370S” amino acid substitutionset. In terms of nomenclature, the pair “S364K/E357Q:L368D/K370S” meansthat one of the monomers includes an Fc domain that includes the aminoacid substitutions S364K and E357Q and the other monomer includes an Fcdomain that includes the amino acid substitutions L368D and K370S; asabove, the “strandedness” of these pairs depends on the starting pI.

In some embodiments, the skew variants provided herein can be optionallyand independently incorporated with any other modifications, including,but not limited to, other skew variants (see, e.g., in FIG. 37 of USPubl. App. No. 2012/0149876, herein incorporated by reference,particularly for its disclosure of skew variants), pI variants,isotpypic variants, FcRn variants, ablation variants, etc. into one orboth of the first and second Fc domains of the heterodimeric antibody.Further, individual modifications can also independently and optionallybe included or excluded from the subject the heterodimeric antibody.

In some embodiments, the skew variants outlined herein can be optionallyand independently incorporated with any pI variant (or other variantssuch as Fc variants, FcRn variants, etc.) into one or both heavy chainmonomers, and can be independently and optionally included or excludedfrom the subject heterodimeric antibodies.

2. pI (Isoelectric point) Variants for Heterodimers

In some embodiments, the heterodimeric antibody includes purificationvariants that advantageously allow for the separation of heterodimericantibody (e.g., anti-B7H3×anti-CD28 bispecific antibody) fromhomodimeric proteins.

There are several basic mechanisms that can lead to ease of purifyingheterodimeric antibodies. For example, modifications to one or both ofthe antibody heavy chain monomers A and B such that each monomer has adifferent pI allows for the isoelectric purification of heterodimericA-B antibody from monomeric A-A and B-B proteins. Alternatively, somescaffold formats, such as the “1+1 Fab-scFv-Fc” format, the “2+1Fab₂-scFv-Fc” format, and the “2+1 CLC” format allows separation on thebasis of size. As described above, it is also possible to “skew” theformation of heterodimers over homodimers using skew variants. Thus, acombination of heterodimerization skew variants and pI variants findparticular use in the heterodimeric antibodies provided herein.

Additionally, as more fully outlined below, depending on the format ofthe heterodimeric antibody, pI variants either contained within theconstant region and/or Fc domains of a monomer, and/or domain linkerscan be used. In some embodiments, the heterodimeric antibody includesadditional modifications for alternative functionalities that can alsocreate pI changes, such as Fc, FcRn and KO variants.

In some embodiments, the subject heterodimeric antibodies providedherein include at least one monomer with one or more modifications thatalter the pI of the monomer (i.e., a “pI variant”). In general, as willbe appreciated by those in the art, there are two general categories ofpI variants: those that increase the pI of the protein (basic changes)and those that decrease the pI of the protein (acidic changes). Asdescribed herein, all combinations of these variants can be done: onemonomer may be wild type, or a variant that does not display asignificantly different pI from wild-type, and the other can be eithermore basic or more acidic. Alternatively, each monomer is changed, oneto more basic and one to more acidic.

Depending on the format of the heterodimer antibody, pI variants can beeither contained within the constant and/or Fc domains of a monomer, orcharged linkers, either domain linkers or scFv linkers, can be used.That is, antibody formats that utilize scFv(s) such as “1+1Fab-scFv-Fc”, format can include charged scFv linkers (either positiveor negative), that give a further pI boost for purification purposes. Aswill be appreciated by those in the art, some 1+1 Fab-scFv-Fc and 2+1Fab₂-scFv-Fc formats are useful with just charged scFv linkers and noadditional pI adjustments, although the invention does provide pIvariants that are on one or both of the monomers, and/or charged domainlinkers as well. In addition, additional amino acid engineering foralternative functionalities may also confer pI changes, such as Fc, FcRnand KO variants.

In subject heterodimeric antibodies that utilizes pI as a separationmechanism to allow the purification of heterodimeric proteins, aminoacid variants are introduced into one or both of the monomerpolypeptides. That is, the pI of one of the monomers (referred to hereinfor simplicity as “monomer A”) can be engineered away from monomer B, orboth monomer A and B change be changed, with the pI of monomer Aincreasing and the pI of monomer B decreasing. As is outlined more fullybelow, the pI changes of either or both monomers can be done by removingor adding a charged residue (e.g., a neutral amino acid is replaced by apositively or negatively charged amino acid residue, e.g., glycine toglutamic acid), changing a charged residue from positive or negative tothe opposite charge (aspartic acid to lysine) or changing a chargedresidue to a neutral residue (e.g., loss of a charge; lysine to serine).A number of these variants are shown in the FIGS. 3 and 4 .

Thus, in some embodiments, the subject heterodimeric antibody includesamino acid modifications in the constant regions that alter theisoelectric point (pI) of at least one, if not both, of the monomers ofa dimeric protein to form “pI antibodies”) by incorporating amino acidsubstitutions (“pI variants” or “pI substitutions”) into one or both ofthe monomers. As shown herein, the separation of the heterodimers fromthe two homodimers can be accomplished if the pIs of the two monomersdiffer by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 orgreater all finding use in the present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the components, for example in the1+1 Fab-scFv-Fc, 2+1 Fab₂-scFv-Fc, 1+1 CLC and 2+1 CLC formats, thestarting pI of the scFv (1+1 Fab-scFv-Fc, 2+1 Fab₂-scFv-Fc) and Fab(s)of interest. That is, to determine which monomer to engineer or in which“direction” (e.g., more positive or more negative), the Fv sequences ofthe two target antigens are calculated and a decision is made fromthere. As is known in the art, different Fvs will have differentstarting pIs which are exploited in the present invention. In general,as outlined herein, the pIs are engineered to result in a total pIdifference of each monomer of at least about 0.1 logs, with 0.2 to 0.5being preferred as outlined herein.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying bispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and pI heterodimerization variants) are notincluded in the variable regions, such that each individual antibodymust be engineered. In addition, in some embodiments, the possibility ofimmunogenicity resulting from the pI variants is significantly reducedby importing pI variants from different IgG isotypes such that pI ischanged without introducing significant immunogenicity. Thus, anadditional problem to be solved is the elucidation of low pI constantdomains with high human sequence content, e.g., the minimization oravoidance of non-human residues at any particular position.Alternatively or in addition to isotypic substitutions, the possibilityof immunogenicity resulting from the pI variants is significantlyreduced by utilizing isosteric substitutions (e.g. Asn to Asp; and Glnto Glu).

As discussed below, a side benefit that can occur with this pIengineering is also the extension of serum half-life and increased FcRnbinding. That is, as described in US Publ. App. No. US 2012/0028304(incorporated by reference in its entirety), lowering the pI of antibodyconstant domains (including those found in antibodies and Fc fusions)can lead to longer serum retention in vivo. These pI variants forincreased serum half-life also facilitate pI changes for purification.

In addition, it should be noted that the pI variants give an additionalbenefit for the analytics and quality control process of bispecificantibodies, as the ability to either eliminate, minimize and distinguishwhen homodimers are present is significant. Similarly, the ability toreliably test the reproducibility of the heterodimeric antibodyproduction is important.

In general, embodiments of particular use rely on sets of variants thatinclude skew variants, which encourage heterodimerization formation overhomodimerization formation, coupled with pI variants, which increase thepI difference between the two monomers to facilitate purification ofheterodimers away from homodimers.

Exemplary combinations of pI variants are shown in FIGS. 4 and 5 , andFIG. 30 of US Publ. App. No. 2016/0355608, all of which are hereinincorporated by reference in its entirety and specifically for thedisclosure of pI variants. Preferred combinations of pI variants areshown in FIGS. 3 and 4 . As outlined herein and shown in the figures,these changes are shown relative to IgG1, but all isotypes can bealtered this way, as well as isotype hybrids. In the case where theheavy chain constant domain is from IgG2-4, R133E and R133Q can also beused.

In one embodiment, a preferred combination of pI variants has onemonomer (the negative Fab side) comprising 208D/295E/384D/418E/421Dvariants (N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) anda second monomer (the positive scFv side) comprising a positivelycharged scFv linker, including (GKPGS)₄ (SEQ ID NO:796). However, aswill be appreciated by those in the art, the first monomer includes aCH1 domain, including position 208. Accordingly, in constructs that donot include a CH1 domain (for example for antibodies that do not utilizea CH1 domain on one of the domains), a preferred negative pI variant Fcset includes 295E/384D/418E/421D variants (Q295E/N384D/Q418E/N421D whenrelative to human IgG1).

Accordingly, in some embodiments, one monomer has a set of substitutionsfrom FIG. 4 and the other monomer has a charged linker (either in theform of a charged scFv linker because that monomer comprises an scFv ora charged domain linker, as the format dictates, which can be selectedfrom those depicted in FIG. 6 ).

In some embodiments, modifications are made in the hinge of the Fcdomain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, and 230 based on EU numbering. Thus, pImutations and particularly substitutions can be made in one or more ofpositions 216-230, with 1, 2, 3, 4 or 5 mutations finding use. Again,all possible combinations are contemplated, alone or with other pIvariants in other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. In some cases, only pI substitutions are done in thehinge domain, and in others, these substitution(s) are added to other pIvariants in other domains in any combination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327,334 and 339, based on EU numbering. It should be noted that changes in233-236 can be made to increase effector function (along with 327A) inthe IgG2 backbone. Again, all possible combinations of these 14positions can be made; e.g., an anti-CD28 or anti-B7H3 antibody providedherein may include a variant Fc domain with 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 CH2 pI substitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, anon-native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non-native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the modifications can be independently andoptionally selected from position 355, 359, 362, 384, 389, 392, 397,418, 419, 444 and 447 (EU numbering) of the CH3 region. Specificsubstitutions that find use in lowering the pI of CH3 domains include,but are not limited to, a non-native glutamine or glutamic acid atposition 355, a non-native serine at position 384, a non-nativeasparagine or glutamic acid at position 392, a non-native methionine atposition 397, a non-native glutamic acid at position 419, a non-nativeglutamic acid at position 359, a non-native glutamic acid at position362, a non-native glutamic acid at position 389, a non-native glutamicacid at position 418, a non-native glutamic acid at position 444, and adeletion or non-native aspartic acid at position 447.

3. Isotypic Variants

In addition, many embodiments of the subject heterodimeric antibodiesrely on the “importation” of pI amino acids at particular positions fromone IgG isotype into another, thus reducing or eliminating thepossibility of unwanted immunogenicity being introduced into thevariants. A number of these are shown in FIG. 21 of US Publ.2014/0370013, hereby incorporated by reference. That is, IgG1 is acommon isotype for therapeutic antibodies for a variety of reasons,including high effector function. However, the heavy constant region ofIgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). Byintroducing IgG2 residues at particular positions into the IgG1backbone, the pI of the resulting monomer is lowered (or increased) andadditionally exhibits longer serum half-life. For example, IgG1 has aglycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI3.22); importing the glutamic acid will affect the pI of the resultingprotein. As is described below, a number of amino acid substitutions aregenerally required to significant affect the pI of the variant antibody.However, it should be noted as discussed below that even changes in IgG2molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g., bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

4. Calculating pI

The pI of each monomer of the antibodies provided herein can depend onthe pI of the variant heavy chain constant domain and the pI of thetotal monomer, including the variant heavy chain constant domain and thefusion partner. Thus, in some embodiments, the change in pI iscalculated on the basis of the variant heavy chain constant domain,using the chart in the FIG. 19 of US Pub. 2014/0370013. As discussedherein, which monomer to engineer is generally decided by the inherentpI of the Fv and scaffold regions. Alternatively, the pI of each monomercan be compared.

5. pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, the pIvariant can have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall' Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half-life as wild-type Fc (Dall' Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

D. Additional Fc Variants for Additional Functionality

In addition to the heterodimerization variants discussed above, thereare a number of useful Fc amino acid modification that can be made for avariety of reasons, including, but not limited to, altering binding toone or more FcγR receptors, altered binding to FcRn receptors, etc, asdiscussed below.

Accordingly, the antibodies provided herein (heterodimeric, as well ashomodimeric) can include such amino acid modifications with or withoutthe heterodimerization variants outlined herein (e.g., the pI variantsand steric variants). Each set of variants can be independently andoptionally included or excluded from any particular heterodimericprotein.

1. FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors. In certainembodiments, the subject antibody includes modifications that alter thebinding to one or more FcγR receptors (i.e., “FcγR variants”).Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto FcγRIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the subject antibodies include those listed in U.S. Pat. No.8,188,321 (particularly FIG. 41) and U.S. Pat. No. 8,084,582, and USPubl. App. Nos. 20060235208 and 20070148170, all of which are expresslyincorporated herein by reference in their entirety and specifically forthe variants disclosed therein that affect Fcγ receptor binding.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and299T. Such modification may be included in one or both Fc domains of thesubject antibody.

In some embodiments, the subject antibody includes one or more Fcmodifications that increase serum half-life. Fc substitutions that finduse in increased binding to the FcRn receptor and increased serumhalf-life, as specifically disclosed in U.S. Ser. No. 12/341,769, herebyincorporated by reference in its entirety, including, but not limitedto, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436Ior V/434S, 436V/428L and 259I/308F/428L. Such modification may beincluded in one or both Fc domains of the subject antibody.

2. Ablation Variants

In some embodiments, the heterodimeric antibody (e.g.,anti-B7H3×anti-CD28 bispecific antibody) includes one or moremodifications that reduce or remove the normal binding of the Fc domainto one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa,FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. Suchmodifications are referred to as “FcγR ablation variants” or “Fc knockout (FcKO or KO)” variants. In these embodiments, for some therapeuticapplications, it is desirable to reduce or remove the normal binding ofthe Fc domain to one or more or all of the Fcγ receptors (e.g., FcγR1,FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms ofaction. That is, for example, in many embodiments, particularly in theuse of bispecific antibodies that bind CD28 monovalently, it isgenerally desirable to ablate FcγRIIIa binding to eliminate orsignificantly reduce ADCC activity. In some embodiments, of the subjectantibodies described herein, at least one of the Fc domains comprisesone or more Fcγ receptor ablation variants. In some embodiments, of thesubject antibodies described herein, both of the Fc domains comprisesone or more Fcγ receptor ablation variants. These ablation variants aredepicted in FIG. 5 , and each can be independently and optionallyincluded or excluded, with preferred aspects utilizing ablation variantsselected from the group consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del. Itshould be noted that the ablation variants referenced herein ablate FcγRbinding but generally not FcRn binding.

As is known in the art, the Fc domain of human IgG1 has the highestbinding to the Fcγ receptors, and thus ablation variants can be usedwhen the constant domain (or Fc domain) in the backbone of theheterodimeric antibody is IgG1. Alternatively, or in addition toablation variants in an IgG1 background, mutations at the glycosylationposition 297 (generally to A or S) can significantly ablate binding toFcγRIIIa, for example. Human IgG2 and IgG4 have naturally reducedbinding to the Fcγ receptors, and thus those backbones can be used withor without the ablation variants.

E. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

Exemplary combination of variants that are included in some embodimentsof the heterodimeric 1+1 Fab-scFv-Fc, 2+1 Fab₂-scFv-Fc, 1+1 CLC and 2+1CLC format antibodies are included in FIG. 8 . In some embodiments, theheterodimeric antibody includes a combination of variants as depicted inFIG. 8 . In certain embodiments, the antibody is a heterodimeric 11+1Fab-scFv-Fc, 2+1 Fab₂-scFv-Fc, 1+1 CLC or 2+1 CLC format antibody.

F. Useful Antibody Formats

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric bispecific antibodies provided herein can takeon several different configurations as generally depicted in FIGS. 33and 34 .

As will be appreciated by those in the art, the heterodimeric formats ofthe invention can have different valencies as well as be bispecific.That is, heterodimeric antibodies of the invention can be bivalent andbispecific, or trivalent and bispecific, wherein the first antigen isbound by two binding domains and the second antigen by a second bindingdomain. As is outlined herein, when CD28 is one of the target antigens,it is preferable that the CD28 is bound only monovalently.

The present invention utilizes CD28 antigen binding domains incombination with B7H3 binding domains. As will be appreciated by thosein the art, any collection of anti-CD28 CDRs, anti-CD28 variable lightand variable heavy domains, Fabs and scFvs as depicted in any of thefigures (see particularly FIGS. 16-21 ) can be used. Similarly, any ofthe anti-B7H3 antigen binding domains can be used, whether CDRs,variable light and variable heavy domains, Fabs and scFvs as depicted inany of the Figures (e.g., FIGS. 29-31 ) can be used, optionally andindependently combined in any combination.

1. 1+1 Fab-scFv-Fc Format (“Bottle Opener”)

One heterodimeric antibody format that finds particular use in subjectbispecific antibodies provided herein (e.g., anti-CD28×anti-B7H3antibody) is the “1+1 Fab-scFv-Fc” or “bottle opener” format as shown inFIG. 33A. The 1+1 Fab-scFv-Fc format antibody includes a first monomerthat is a “regular” heavy chain (VH1-CH1-hinge-CH2-CH3), wherein VH1 isa first variable heavy domain and CH2-CH3 is a first Fc domain. The 1+1Fab-scFv-Fc also includes a light chain that includes a first variablelight domain VL1 and a constant light domain CL. The light chaininteracts with the VH1-CH1 of the first monomer to form a first antigenbinding domain that is a Fab. The second monomer of the antibodyincludes a second binding domain that is a single chain Fv (“scFv”, asdefined below) and a second Fc domain. The scFv includes a secondvariable heavy domain (VH2) and a second variable light domain (VL2),wherein the VH2 is attached to the VL2 using an scFv linker that can becharged (see, e.g., FIG. 6 ). The scFv is attached to the heavy chainusing a domain linker (see, e.g., FIG. 7 ). The two monomers are broughttogether by the use of amino acid variants (e.g., heterodimerizationvariants, discussed above) in the constant regions (e.g., the Fc domain,the CH1 domain and/or the hinge region) that promote the formation ofheterodimeric antibodies as is described more fully below. Thisstructure is sometimes referred to herein as the “bottle-opener” format,due to a rough visual similarity to a bottle-opener. In someembodiments, the 1+1 Fab-scFv-Fc format antibody is a bivalent antibody.

There are several distinct advantages to the present “1+1 Fab-scFv-Fc”format. As is known in the art, antibody analogs relying on two scFvconstructs often have stability and aggregation problems, which can bealleviated in the present invention by the addition of a “regular” heavyand light chain pairing. In addition, as opposed to formats that rely ontwo heavy chains and two light chains, there is no issue with theincorrect pairing of heavy and light chains (e.g., heavy 1 pairing withlight 2, etc.).

In some embodiments of the 1+1 Fab-scFv-Fc format antibody, one of thefirst or second antigen binding domain is a CD28 binding domain and theother binding domain is a tumor associated antigen (TAA) binding domain.In some embodiments where the 1+1 Fab-scFv-Fc includes a CD28 bindingdomain and a tumor associated antigen (TAA) binding domain, it is thescFv that binds to the CD28, and the Fab that binds the TAA. In someembodiments, the TAA is B7H3. Exemplary anti-B7H3×anti-CD28 bispecificantibodies in the 1+1 Fab-scFv-Fc format is depicted in FIG. 35 .

In some embodiments, the first and second Fc domains of the 1+1Fab-scFv-Fc format antibody are variant Fc domains that includeheterodimerization skew variants (e.g., a set of amino acidsubstitutions as shown in FIGS. 3 and 9 ). Particularly usefulheterodimerization skew variants include S364K/E357Q:L368D/K370S;L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K;L368D/K370S: S364K/E357L; K370S: S364K/E357Q; T366S/L368A/Y407V: T366Wand T366S/L368A/Y407V/Y349C: T366W/S354C (EU numbering)). In exemplaryembodiments, one of the first or second variant Fc domains includesheterodimerization skew variants L368D/K370S and the other of the firstor second variant Fc domains includes heterodimerization skew variantsS364K/E357Q, wherein numbering is according to EU numbering. Inexemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q, whereinnumbering is according to EU numbering.

In some embodiments, the variant Fc domains include ablation variants(including those shown in FIG. 5 ). In some embodiments, each of thefirst and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K, wherein numbering is according to EUnumbering.

In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst monomer includes pI variants (including those shown in FIG. 4 ).In exemplary embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst monomer includes pI variants N208D/Q295E/N384D/Q418E/N421D,wherein numbering is according to EU numbering.

In exemplary embodiments, the CH1-hinge-CH2-CH3 of the first monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,the second Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, the scFv of the 1+1 Fab-scFv-Fc format antibodyprovided herein includes a charged scFv linker (including those shown inFIG. 6 ). In some embodiments, the 1+1 Fab-scFv-Fc format antibodyprovided herein includes FcRn variants M428L/N434S, wherein numbering isaccording to EU numbering.

In exemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q; each ofthe first and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K; and the constant domain(CH1-hinge-CH2-CH3) of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering. In some embodiments, the scFv of the 1+1 Fab-scFv-Fc formatantibody provided herein includes a (GKPGS)₄ charged scFv linker. Insome embodiments, the 1+1 Fab-scFv-Fc format antibody provided hereinincludes FcRn variants M428L/N434S, wherein numbering is according to EUnumbering.

In some embodiments, one of the first binding domain or the secondbinding domain binds CD28 and the other binding domain binds a tumorassociated antigen (TAA) (see FIG. 34A). Any suitable CD28 bindingdomain can be included in subject 1+1 Fab-scFv-Fc format antibody,including any of the CD28 binding domains provided herein. In someembodiments, the CD28 binding domain is one of the following CD28binding domains or a variant thereof: 1A7[CD28]_H1.14L1,1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71,CD28.3[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23 and Sequence Listing).

In some embodiments of the mAb-scFv format, the anti-CD28 ABD has a VHdomain with an amino acid sequence selected from the group consisting ofSEQ ID NO: 870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ IDNO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597,SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ IDNO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611,SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ IDNO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198,SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ IDNO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637,SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ IDNO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651,SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ IDNO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQID NO:671 and SEQ ID NO:672, and a VL domain with an amino acid sequenceselected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657,SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ IDNO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671,SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ IDNO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685,SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ IDNO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699,SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ IDNO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713,SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ IDNO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727,SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ IDNO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741,SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ IDNO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755,SEQ ID NO:1200 and SEQ ID NO:756.

In some embodiments, one of the first binding domain or the secondbinding domain of the 1+1 Fab-scFv-Fc format antibody binds a tumorassociated antigen (TAA). Suitable TAAs include any of the TAAsdisclosed herein. In exemplary embodiments, the TAA is B7H3. Anysuitable B7H3 binding domain can be included in subject 1+1 Fab-scFv-Fcformat antibody, including any of the B7H3 binding domains providedherein. In some embodiments, the B7H3 binding domain is one of thefollowing B7H3 binding domains or a variant thereof:2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704(FIGS. 26-31 and the Sequence Listing).

In some embodiments, the anti-B7H3 ABD has a VL domain with an aminoacid sequence selected from the group consisting of a variable heavydomain with an amino acid sequence selected from the group consisting ofSEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ IDNO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508,SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ IDNO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523,SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ IDNO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537,SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ IDNO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551,SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ IDNO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565,SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ IDNO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579,SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583 and SEQ IDNO:584; and a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO:874 and SEQ ID NO: 932.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:946; and a variable light domain havingthe amino acid sequence of SEQ ID NO:950.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:956; and a variable light domain havingthe amino acid sequence of SEQ ID NO:960.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:964; and a variable light domain havingthe amino acid sequence of SEQ ID NO:968.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:972; and a variable light domain havingthe amino acid sequence of SEQ ID NO:976.

In some embodiments, the 1+1 Fab-scFv-Fc format antibody includes afirst binding domain that binds CD28 and a second binding domain thatbinds B7H3. In some embodiments, the CD28 binding domain is one of thefollowing CD28 binding domains or a variant thereof: 1A7[CD28]_H1.14L1,1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71,CD28.3[CD28]_H0L0, TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1,281VL4[CD28]_H1L1, HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0,hu9.3[CD28]_H1L1 (FIGS. 18-21 and 23 and Sequence Listing).

In some embodiments, the B7H3 binding domain is one of the followingB7H3 binding domains or a variant thereof: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, and m1704 (FIGS. 26-31 and theSequence Listing).

In some embodiments, the anti-B7H3 ABD has a VH domain and VL domainwith amino acid sequences selected from the pairs of a) SEQ ID NOs: 89and 93 from omburamab, b) SEQ ID NOs:97 and 101 from enoblituzumab, c)SEQ ID NOs:105 and 109 from BRCA84D, d) SEQ ID NOs:113 and 117 fromBRCA69D, e) SEQ ID NOs:121 and 125 from PRCA157, f) SEQ ID NOs:129 and133 from huPRCA157, g) SEQ ID NOs:137 and 141 from Mab-D; h) SEQ IDNOs:145 and 149 humAb-D; i) SEQ ID NOs:153 and 157 from m30; j) SEQ IDNOs:161 and 165 from M30-H1-L4, k) SEQ ID NOs:169 and 173 SP265; l) SEQID NOs:177 and 181 from S10-H50L58; m) SEQ ID NOs:185 and 189 from 8H9,n) SEQ ID NOs:193 and 197 from m852; o) SEQ ID NOs:201 and 205 fromm857; p) SEQ ID NOs:209 and 213 from m8524; q) SEQ ID NOs:217 and 221from 1-1; r) SEQ ID NOs:225 and 229 from 1-2; s) SEQ ID NOs:233 and 237from 1-4; t) SEQ ID NOs:241 and 245 from 1-5; u) SEQ ID NOs:249 and 253from 1-7; v) SEQ ID NOs:257 and 261 from 2-5; w) SEQ ID NOs:265 and 269from 2-8; x) SEQ ID NOs: 273 and 277 from chAb2; y) SEQ ID NOs:281 and285 chAb3; z) SEQ ID NOs:289 and 293 from chAb4; aa) SEQ ID NOs:297 and301 from chAb18; bb) SEQ ID NOs:305 and 309 from chAb13; cc) SEQ IDNOs:313 and 317 from chAb12; dd) SEQ ID NOs:321 and 325 from chAb14; ee)SEQ ID NOs:329 and 333 from chAb6; ff) SEQ ID NOs:337 and 341 fromchAb11, gg) SEQ ID NOs:345 and 349 from chAB16; hh) SEQ ID NOs:353 and357 from chAb10; ii) SEQ ID NOs:361 and 365 from ChAb7; jj) SEQ IDNOs:369 and 373 from chAb8, kk) SEQ ID NOs:377 and 381 from chAb17; ll)SEQ ID NOs:385 and 389 from chAb5, mm) SEQ ID NOs:393 and 397 fromhuAb3v2.5, nn) SEQ ID NOs:401 and 405 from huAb3v2.6, pp) SEQ ID NOs:409and 413 from huAb13v1, qq) SEQ ID NOs:417 and 421 from TPP-5706, rr) SEQID NOs:425 and 429 from TPP-6642; ss) SEQ ID NOs:433 and 437 fromTPP-6850, tt) SEQ ID NOs:441 and 445 from TPP-3803, uu) SEQ ID NOs:449and 453 from TRL4542, vv) SEQ ID NOs:457 and 461 from h1702, ww) SEQ IDNOs:465 and 469 from h1703, xx) SEQ ID NOs:473 and 477 from huA3, yy)SEQ ID NOs:481 and 485 from huA9 and zz) SEQ ID NOs: 489 and 493 fromm1704. See FIG. 17 from U.S. Ser. No. 63/092,272.

FIG. 10 shows some exemplary Fc domain sequences that are useful in the1+1 Fab-scFv-Fc format antibodies. The “monomer 1” sequences depicted inFIG. 10 typically refer to the Fc domain of the “Fab-Fc heavy chain” andthe “monomer 2” sequences refer to the Fc domain of the “scFv-Fc heavychain.” In addition, FIGS. 12-15 provides exemplary CH1-hinge domains,CH1 domains, and hinge domains that can be included in the first orsecond monomer of the 1+1 Fab-scFv-Fc format. Further, FIG. 16 providesuseful CL sequences that can be used with this format.

2. 2+1 Fab₂-scFv-Fc Format (Central-scFv Format)

One heterodimeric antibody format that finds particular use in thesubject bispecific antibodies provided herein (e.g., anti-CD28×anti-B7H3antibody) is the 2+1 Fab₂-scFv-Fc format (also referred to as“central-scFv format”) shown in FIG. 33B. This antibody format includesthree antigen binding domains: two Fab portions and an scFv that isinserted between the VH—CH1 and CH2-CH3 regions of one of the monomers.In some embodiments of this format, the Fab portions each bind a tumorassociated antigen (TAA) and the “extra” scFv domain binds CD28. In someembodiments, the 2+1 Fab₂-scFv-Fc format antibody is a trivalentantibody.

In some embodiments of the 2+1 Fab₂-scFv-Fc format, a first monomerincludes a standard heavy chain (i.e., VH1-CH1-hinge-CH2-CH3), whereinVH1 is a first variable heavy domain and CH2-CH3 is a first Fc domain. Asecond monomer includes another first variable heavy domain (VH1), a CH1domain (and optional hinge), a second Fc domain, and an scFv thatincludes an scFv variable light domain (VL2), an scFv linker and a scFvvariable heavy domain (VH2). The scFv is covalently attached between theC-terminus of the CH1 domain of the second monomer and the N-terminus ofthe second Fc domain using optional domain linkers (VH1-CH1-[optionallinker]-VH2-scFv linker-VH2-[optional linker]-CH2-CH3, or the oppositeorientation for the scFv, VH1-CH1-[optional linker]-VL2-scFvlinker-VH2-[optional linker]-CH2-CH3). The optional linkers can be anysuitable peptide linkers, including, for example, the domain linkersincluded in FIG. 7 . This embodiment further utilizes a common lightchain that includes a variable light domain (VL1) and a constant lightdomain (CL). The common light chain associates with the VH1-CH1 of thefirst and second monomers to form two identical Fabs. In someembodiments, the identical Fabs each bind a tumor associated antigen(e.g., B7H3). As for many of the embodiments herein, these constructscan include skew variants, pI variants, ablation variants, additional Fcvariants, etc. as desired and described herein.

In some embodiments, the first and second Fc domains of the 2+1Fab₂-scFv-Fc format antibody are variant Fc domains that includeheterodimerization skew variants (e.g., a set of amino acidsubstitutions as shown in FIGS. 3 and 9 ). Particularly usefulheterodimerization skew variants include S364K/E357Q:L368D/K370S;L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K;L368D/K370S: S364K/E357L; K370S: S364K/E357Q; T366S/L368A/Y407V: T366Wand T366S/L368A/Y407V/Y349C: T366W/S354C (EU numbering)). In exemplaryembodiments, one of the first or second variant Fc domains includesheterodimerization skew variants L368D/K370S and the other of the firstor second variant Fc domains includes heterodimerization skew variantsS364K/E357Q, wherein numbering is according to EU numbering. Inexemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q, whereinnumbering is according to EU numbering.

In some embodiments, the variant Fc domains include ablation variants(including those shown in FIG. 5 ). In some embodiments, each of thefirst and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K, wherein numbering is according to EUnumbering.

In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst monomer includes pI variants (including those shown in FIG. 4 ).In exemplary embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst monomer includes pI variants N208D/Q295E/N384D/Q418E/N421D,wherein numbering is according to EU numbering.

In some embodiments, the scFv of the 2+1 Fab₂-scFv-Fc format antibodyprovided herein includes a charged scFv linker (including those shown inFIG. 6 ). In some embodiments, the 2+1 Fab₂-scFv-Fc format antibodyprovided herein includes FcRn variants M428L/N434S, wherein numbering isaccording to EU numbering.

In exemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q; each ofthe first and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K; and the constant domain(CH1-hinge-CH2-CH3) of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering. In some embodiments, the scFv of the 2+1 Fab₂-scFv-Fc formatantibody provided herein includes a (GKPGS)₄ charged scFv linker. Insome embodiments, the 2+1 Fab₂-scFv-Fc format antibody provided hereinincludes FcRn variants M428L/N434S, wherein numbering is according to EUnumbering.

In some embodiments, the CH1-hinge-CH2-CH3 of the first monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the second Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, the scFv of the second monomer of the 2+1Fab₂-scFv-Fc format antibody is a CD28 binding and the VH1 of the firstand second monomer and the VL1 of the common light chain each formbinding domains that bind a tumor associated antigen (TAA, e.g., B7H3)(see FIG. 34B). Any suitable CD28 binding domain can be included insubject 2+1 Fab₂-scFv-Fc format antibody, including any of the CD28binding domains provided herein. In some embodiments, the CD28 bindingdomain is one of the following CD28 binding domains or a variantthereof: 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, TGN1412_H1L1,341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1(FIGS. 18-21 and 23 and the Sequence Listing). In some embodiments ofthe mAb-scFv format, the anti-CD28 ABD has a VH domain with an aminoacid sequence selected from the group consisting of SEQ ID NO: 870, SEQID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ ID NO:588, SEQ ID NO:589,SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQ ID NO:593, SEQ IDNO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597, SEQ ID NO:598, SEQID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ ID NO:602, SEQ ID NO:603,SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQ ID NO:607, SEQ IDNO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611, SEQ ID NO:612, SEQID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ ID NO:616, SEQ ID NO:617,SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQ ID NO:621, SEQ IDNO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198, SEQ ID NO:1199,SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ ID NO:628, SEQ IDNO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQ ID NO:633, SEQID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637, SEQ ID NO:638,SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ ID NO:642, SEQ IDNO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQ ID NO:647, SEQID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651, SEQ ID NO:652,SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ IDNO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQ ID NO:671 andSEQ ID NO:672, and a VL domain with an amino acid sequence selected fromthe group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQ ID NO:653, SEQID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658,SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ ID NO:662, SEQ IDNO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQ ID NO:667, SEQID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671, SEQ ID NO:672,SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ ID NO:676, SEQ IDNO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQ ID NO:681, SEQID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685, SEQ ID NO:686,SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ ID NO:690, SEQ IDNO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQ ID NO:695, SEQID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699, SEQ ID NO:700,SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ ID NO:704, SEQ IDNO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQ ID NO:709, SEQID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713, SEQ ID NO:714,SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ ID NO:718, SEQ IDNO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQ ID NO:723, SEQID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727, SEQ ID NO:728,SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ ID NO:732, SEQ IDNO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQ ID NO:737, SEQID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741, SEQ ID NO:742,SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ ID NO:746, SEQ IDNO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQ ID NO:751, SEQID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755, SEQ ID NO:1200and SEQ ID NO:756.

In some embodiments, the VH1 of the first and second monomer and the VL1of the common light chain of the 2+1 Fab₂-scFv-Fc format antibody eachform a binding domain that binds a tumor associated antigen (TAA) (seeFIG. 34B). Suitable TAAs include any of the TAAs disclosed herein. Inexemplary embodiments, the TAA is B7H3. Any suitable B7H3 binding domaincan be included in subject 2+1 Fab₂-scFv-Fc format antibody, includingany of the B7H3 binding domains provided herein. In some embodiments,the B7H3 binding domain is one of the following B7H3 binding domains ora variant thereof: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, m1704 (FIGS. 26-31 and the SequenceListing). In some embodiments, the anti-B7H3 ABD has a VH domain and VLdomain with amino acid sequences selected from the pairs of a) SEQ IDNOs: 89 and 93 from omburamab, b) SEQ ID NOs:97 and 101 fromenoblituzumab, c) SEQ ID NOs:105 and 109 from BRCA84D, d) SEQ ID NOs:113and 117 from BRCA69D, e) SEQ ID NOs:121 and 125 from PRCA157, f) SEQ IDNOs:129 and 133 from huPRCA157, g) SEQ ID NOs:137 and 141 from Mab-D; h)SEQ ID NOs:145 and 149 humAb-D; i) SEQ ID NOs:153 and 157 from m30; j)SEQ ID NOs:161 and 165 from M30-H1-L4, k) SEQ ID NOs:169 and 173 SP265;l) SEQ ID NOs:177 and 181 from S10-H50L58; m) SEQ ID NOs:185 and 189from 8H9, n) SEQ ID NOs:193 and 197 from m852; o) SEQ ID NOs:201 and 205from m857; p) SEQ ID NOs:209 and 213 from m8524; q) SEQ ID NOs:217 and221 from 1-1; r) SEQ ID NOs:225 and 229 from 1-2; s) SEQ ID NOs:233 and237 from 1-4; t) SEQ ID NOs:241 and 245 from 1-5; u) SEQ ID NOs:249 and253 from 1-7; v) SEQ ID NOs:257 and 261 from 2-5; w) SEQ ID NOs:265 and269 from 2-8; x) SEQ ID NOs: 273 and 277 from chAb2; y) SEQ ID NOs:281and 285 chAb3; z) SEQ ID NOs:289 and 293 from chAb4; aa) SEQ ID NOs:297and 301 from chAb18; bb) SEQ ID NOs:305 and 309 from chAb13; cc) SEQ IDNOs:313 and 317 from chAb12; dd) SEQ ID NOs:321 and 325 from chAb14; ee)SEQ ID NOs:329 and 333 from chAb6; ff) SEQ ID NOs:337 and 341 fromchAb11, gg) SEQ ID NOs:345 and 349 from chAB16; hh) SEQ ID NOs:353 and357 from chAb10; ii) SEQ ID NOs:361 and 365 from ChAb7; jj) SEQ IDNOs:369 and 373 from chAb8, kk) SEQ ID NOs:377 and 381 from chAb17; ll)SEQ ID NOs:385 and 389 from chAb5, mm) SEQ ID NOs:393 and 397 fromhuAb3v2.5, nn) SEQ ID NOs:401 and 405 from huAb3v2.6, pp) SEQ ID NOs:409and 413 from huAb13v1, qq) SEQ ID NOs:417 and 421 from TPP-5706, rr) SEQID NOs:425 and 429 from TPP-6642; ss) SEQ ID NOs:433 and 437 fromTPP-6850, tt) SEQ ID NOs:441 and 445 from TPP-3803, uu) SEQ ID NOs:449and 453 from TRL4542, vv) SEQ ID NOs:457 and 461 from h1702, ww) SEQ IDNOs:465 and 469 from h1703, xx) SEQ ID NOs:473 and 477 from huA3, yy)SEQ ID NOs:481 and 485 from huA9 and zz) SEQ ID NOs: 489 and 493 fromm1704. See FIG. 17 from U.S. Ser. No. 63/092,272.

In some embodiments, the anti-B7H3 ABD has a VL domain with an aminoacid sequence selected from the group consisting of a variable heavydomain with an amino acid sequence selected from the group consisting ofSEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ IDNO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508,SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ IDNO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523,SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ IDNO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537,SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ IDNO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551,SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ IDNO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565,SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ IDNO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579,SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583 and SEQ IDNO:584; and a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO:874 and SEQ ID NO: 932.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:946; and a variable light domain havingthe amino acid sequence of SEQ ID NO:950.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:956; and a variable light domain havingthe amino acid sequence of SEQ ID NO:960.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:964; and a variable light domain havingthe amino acid sequence of SEQ ID NO:968.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:972; and a variable light domain havingthe amino acid sequence of SEQ ID NO:976.

FIG. 11 shows some exemplary Fc domain sequences that are useful withthe 2+1 Fab₂-scFv-Fc format. The “monomer 1” sequences depicted in FIG.11 typically refer to the Fc domain of the “Fab-Fc heavy chain” and the“monomer 2” sequences refer to the Fc domain of the “Fab-scFv-Fc heavychain.” In addition, FIGS. 12-15 provides exemplary CH1-hinge domains,CH1 domains, and hinge domains that can be included in the first orsecond monomer of the 2+1 Fab₂-scFv-Fc format. Further, FIG. 16 providesuseful CL sequences that can be used with this format. Exemplaryanti-B7H3×anti-CD28 bispecific antibodies in the 2+1 Fab₂-scFv-Fc formatare depicted in FIG. 36 .

3. 1+1 CLC Format

One heterodimeric antibody format that finds particular use in subjectbispecific antibodies provided herein (e.g., anti-CD28×anti-B7H3antibody) is the “1+1 Common Light Chain” or “1+1 CLC” format, which isdepicted in FIG. 33C. The 1+1 CLC format antibody includes a firstmonomer that includes a VH1-CH1-hinge-CH2-CH3, wherein VH1 is a firstvariable heavy domain and CH2-CH3 is a first Fc domain; a second monomerthat includes a VH2-CH1-hinge-CH2-CH3, wherein VH2 is a second variableheavy domain and CH2-C3 is a second Fc domain; and a third monomer“common light chain” comprising VL-CL, wherein VL is a common variablelight domain and CL is a constant light domain. In such embodiments, theVL pairs with the VH1 to form a first binding domain with a firstantigen binding specificity; and the VL pairs with the VH2 to form asecond binding domain with a second antigen binding specificity. In someembodiments, the 1+1 CLC format antibody is a bivalent antibody.

In some embodiments, the first and second Fc domains of the 1+1 CLCformat are variant Fc domains that include heterodimerization skewvariants (e.g., a set of amino acid substitutions as shown in FIGS. 3and 9 ). Particularly useful heterodimerization skew variants includeS364K/E357Q:L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K;T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L; K370S: S364K/E357Q;T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C (EUnumbering). In exemplary embodiments, one of the first or second variantFc domains includes heterodimerization skew variants L368D/K370S and theother of the first or second variant Fc domains includesheterodimerization skew variants S364K/E357Q, wherein numbering isaccording to EU numbering. In exemplary embodiments, the first variantFc domain includes heterodimerization skew variants L368D/K370S and thesecond variant Fc domain includes heterodimerization skew variantsS364K/E357Q, wherein numbering is according to EU numbering.

In some embodiments, the variant Fc domains include ablation variants(including those shown in FIG. 5 ). In some embodiments, each of thefirst and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K, wherein numbering is according to EUnumbering.

In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst or second monomer includes pI variants (including those shown inFIG. 4 ). In exemplary embodiments, the constant domain(CH1-hinge-CH2-CH3) of the first or second monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In some embodiments, the 1+1 CLC format antibody provided hereinincludes FcRn variants M428L/N434S, wherein numbering is according to EUnumbering.

In exemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q; each ofthe first and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K; and the constant domain(CH1-hinge-CH2-CH3) of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In some embodiments, the CH1-hinge-CH2-CH3 of the first monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the second Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, the 1+1 CLC format antibody provided herein furtherincludes FcRn variants M428L/N434S, wherein numbering is according to EUnumbering.

In some embodiments, one of the first binding domain or the secondbinding domain binds CD28 and the other binding domain binds a tumorassociated antigen (TAA) (see FIG. 34C). Any suitable CD28 bindingdomain can be included in subject 1+1 CLC format antibody, including anyof the CD28 binding domains provided herein. In some embodiments, theCD28 binding domain is one of the following CD28 binding domains or avariant thereof: 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1(FIGS. 18-21 and 23 and Sequence Listing). In exemplary embodiments, theCD28 binding domain includes a 1A7[CD28]_H1.14 variable heavy domain. Insome embodiments, the CD28 binding domain includes a 1A7[CD28]_H1.14variable heavy domain or variant thereof and a light variable domain ofany of the CD28 binding domains provided herein. In exemplaryembodiments, the CD28 binding domain is 1A7[CD28]_H1.14L1 or a variantthereof. In some embodiments of the mAb-scFv format, the anti-CD28 ABDhas a VH domain with an amino acid sequence selected from the groupconsisting of SEQ ID NO: 870, SEQ ID NO:585, SEQ ID NO:586, SEQ IDNO:587, SEQ ID NO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQID NO:592, SEQ ID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596,SEQ ID NO:597, SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ IDNO:601, SEQ ID NO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQID NO:606, SEQ ID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610,SEQ ID NO:611, SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ IDNO:615, SEQ ID NO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQID NO:620, SEQ ID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624,SEQ ID NO:1198, SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ IDNO:627, SEQ ID NO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQID NO:632, SEQ ID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636,SEQ ID NO:637, SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ IDNO:641, SEQ ID NO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQID NO:646, SEQ ID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650,SEQ ID NO:651, SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ IDNO:655, SEQ ID NO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQID NO:670, SEQ ID NO:671 and SEQ ID NO:672, and a VL domain with anamino acid sequence selected from the group consisting of SEQ ID NO:874,SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ IDNO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQID NO:661, SEQ ID NO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665,SEQ ID NO:666, SEQ ID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ IDNO:670, SEQ ID NO:671, SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQID NO:675, SEQ ID NO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679,SEQ ID NO:680, SEQ ID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ IDNO:684, SEQ ID NO:685, SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQID NO:689, SEQ ID NO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693,SEQ ID NO:694, SEQ ID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ IDNO:698, SEQ ID NO:699, SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQID NO:703, SEQ ID NO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707,SEQ ID NO:708, SEQ ID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ IDNO:712, SEQ ID NO:713, SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQID NO:717, SEQ ID NO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721,SEQ ID NO:722, SEQ ID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ IDNO:726, SEQ ID NO:727, SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQID NO:731, SEQ ID NO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735,SEQ ID NO:736, SEQ ID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ IDNO:740, SEQ ID NO:741, SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQID NO:745, SEQ ID NO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749,SEQ ID NO:750, SEQ ID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ IDNO:754, SEQ ID NO:755, SEQ ID NO:1200 and SEQ ID NO:756.

In some embodiments, one of the first binding domain or the secondbinding domain of the 1+1 CLC format antibody binds a tumor associatedantigen (TAA). Suitable TAAs include any of the TAAs disclosed herein.In exemplary embodiments, the TAA is B7H3. Any suitable B7H3 bindingdomain can be included in subject 1+1 CLC format antibody, including anyof the B7H3 binding domains provided herein. In some embodiments, theB7H3 binding domain is one of the following B7H3 binding domains or avariant thereof: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, omburtamab, enoblituzumab, BRCA84D, BRCA69D, PRCA157,huPRCA157, mAb-D, humAb-D, M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852,m857, m8524, 1-1, 1-2, 1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4,chAb18, chAb13, chAb12, chAb14, chAb6, chAb11, chAb16, chAb10, chAb7,chAb8, chAb17, chAb5, huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706,TPP-6642, TPP-6850, TPP-3803, TRL4542, h1702, h1703, huA3, huA9, m1704(FIGS. 26-31 and the Sequence Listing).

In some embodiments, the anti-B7H3 ABD has a VL domain with an aminoacid sequence selected from the group consisting of a variable heavydomain with an amino acid sequence selected from the group consisting ofSEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ IDNO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508,SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ IDNO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523,SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ IDNO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537,SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ IDNO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551,SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ IDNO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565,SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ IDNO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579,SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583 and SEQ IDNO:584; and a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO:874 and SEQ ID NO: 932.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:946; and a variable light domain havingthe amino acid sequence of SEQ ID NO:950.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:956; and a variable light domain havingthe amino acid sequence of SEQ ID NO:960.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:964; and a variable light domain havingthe amino acid sequence of SEQ ID NO:968.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:972; and a variable light domain havingthe amino acid sequence of SEQ ID NO:976.

In some embodiments, the anti-B7H3 ABD has a VH domain and VL domainwith amino acid sequences selected from the pairs of a) SEQ ID NOs: 89and 93 from omburamab, b) SEQ ID NOs:97 and 101 from enoblituzumab, c)SEQ ID NOs:105 and 109 from BRCA84D, d) SEQ ID NOs:113 and 117 fromBRCA69D, e) SEQ ID NOs:121 and 125 from PRCA157, f) SEQ ID NOs:129 and133 from huPRCA157, g) SEQ ID NOs:137 and 141 from Mab-D; h) SEQ IDNOs:145 and 149 humAb-D; i) SEQ ID NOs:153 and 157 from m30; j) SEQ IDNOs:161 and 165 from M30-H1-L4, k) SEQ ID NOs:169 and 173 SP265; l) SEQID NOs:177 and 181 from S10-H50L58; m) SEQ ID NOs:185 and 189 from 8H9,n) SEQ ID NOs:193 and 197 from m852; o) SEQ ID NOs:201 and 205 fromm857; p) SEQ ID NOs:209 and 213 from m8524; q) SEQ ID NOs:217 and 221from 1-1; r) SEQ ID NOs:225 and 229 from 1-2; s) SEQ ID NOs:233 and 237from 1-4; t) SEQ ID NOs:241 and 245 from 1-5; u) SEQ ID NOs:249 and 253from 1-7; v) SEQ ID NOs:257 and 261 from 2-5; w) SEQ ID NOs:265 and 269from 2-8; x) SEQ ID NOs: 273 and 277 from chAb2; y) SEQ ID NOs:281 and285 chAb3; z) SEQ ID NOs:289 and 293 from chAb4; aa) SEQ ID NOs:297 and301 from chAb18; bb) SEQ ID NOs:305 and 309 from chAb13; cc) SEQ IDNOs:313 and 317 from chAb12; dd) SEQ ID NOs:321 and 325 from chAb14; ee)SEQ ID NOs:329 and 333 from chAb6; ff) SEQ ID NOs:337 and 341 fromchAb11, gg) SEQ ID NOs:345 and 349 from chAB16; hh) SEQ ID NOs:353 and357 from chAb10; ii) SEQ ID NOs:361 and 365 from ChAb7; jj) SEQ IDNOs:369 and 373 from chAb8, kk) SEQ ID NOs:377 and 381 from chAb17; ll)SEQ ID NOs:385 and 389 from chAb5, mm) SEQ ID NOs:393 and 397 fromhuAb3v2.5, nn) SEQ ID NOs:401 and 405 from huAb3v2.6, pp) SEQ ID NOs:409and 413 from huAb13v1, qq) SEQ ID NOs:417 and 421 from TPP-5706, rr) SEQID NOs:425 and 429 from TPP-6642; ss) SEQ ID NOs:433 and 437 fromTPP-6850, tt) SEQ ID NOs:441 and 445 from TPP-3803, uu) SEQ ID NOs:449and 453 from TRL4542, vv) SEQ ID NOs:457 and 461 from h1702, ww) SEQ IDNOs:465 and 469 from h1703, xx) SEQ ID NOs:473 and 477 from huA3, yy)SEQ ID NOs:481 and 485 from huA9 and zz) SEQ ID NOs: 489 and 493 fromm1704. See FIG. 17 from U.S. Ser. No. 63/092,272.

In exemplary embodiments, the B7H3 binding domain includes a1A7[CD28]_H1.14 variable heavy domain. In some embodiments, the B7H3binding domain includes a 2E4A3.189[B7H3]_H1.22 variable heavy domainand a light variable domain of any of the CD28 or B7H3 binding domainsprovided herein. In exemplary embodiments, the B7H3 binding domainincludes a 2E4A3.189[B7H3]_H1.22 variable heavy domain or a variantthereof and a 1A7[CD28]_L1 variable light domain or a variant thereof.

In some embodiments, the 1+1 CLC format antibody includes a firstbinding domain that binds CD28 and a second binding domain that bindsB7H3. In particular embodiments, the variable heavy domain of the firstbinding domain (i.e., the CD28 binding domain) is a 1A7[CD28]_H1.14variable heavy domain or variant thereof. In some embodiments, thevariable heavy domain of the second binding domain (i.e., the B7H3binding domain) is a 2E4A3.189[B7H3]_H1.22 variable heavy domain orvariant thereof. In some embodiments, the 1+1 CLC format antibodyincludes a common light chain that includes the variable light domain ofany of the CD28 or B7H3 binding domains provided herein. In someembodiments, the variable light domain is a 1A7[CD28]_L1 variable lightdomain or a variant thereof. Exemplary anti-B7H3×anti-CD28 bispecificantibodies in the 1+1 CLC format are depicted in FIG. 37 .

4. 2+1 CLC Format

Another heterodimeric antibody format that finds particular use insubject bispecific antibodies provided herein (e.g., anti-CD28×anti-B7H3antibody) is the “2+1 Common Light Chain” or “2+1 CLC” format, which isdepicted in FIG. 33D. The 2+1 CLC format includes a first monomer thatincludes a VH1-CH1-linker-VH1-CH1-hinge-CH2-CH3, wherein the VH1s areeach a first variable heavy domain and CH2-CH3 is a first Fc domain; asecond monomer that includes a VH2-CH1-hinge-CH2-CH3, wherein VH2 is asecond variable heavy domain and CH2-CH3 is a second Fc domain; and athird monomer that includes a “common light chain” VL-CL, wherein VL isa common variable light domain and CL is a constant light domain. The VLpairs with each of the VH1s of the first monomer to form two firstbinding domains, each with a first antigen binding specificity; and theVL pairs with the VH2 to form a second binding domain with a secondantigen binding specificity. The linker of the first monomer can be anysuitable linker, including any one of the domain linkers or combinationsthereof described in FIG. 7 . In some embodiments, the linker isEPKSCGKPGSGKPGS (SEQ ID NO:1182). In some embodiments, the 2+1 CLCformat antibody is a trivalent antibody.

In some embodiments, the first and second Fc domains of the 2+1 CLCformat are variant Fc domains that include heterodimerization skewvariants (e.g., a set of amino acid substitutions as shown in FIGS. 3and 9 ). Particularly useful heterodimerization skew variants includeS364K/E357Q:L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K;T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L; K370S: S364K/E357Q;T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C (EUnumbering)). In exemplary embodiments, one of the first or secondvariant Fc domains includes heterodimerization skew variants L368D/K370Sand the other of the first or second variant Fc domains includesheterodimerization skew variants S364K/E357Q, wherein numbering isaccording to EU numbering. In exemplary embodiments, the first variantFc domain includes heterodimerization skew variants L368D/K370S and thesecond variant Fc domain includes heterodimerization skew variantsS364K/E357Q, wherein numbering is according to EU numbering.

In some embodiments, the variant Fc domains include ablation variants(including those shown in FIG. 5 ). In some embodiments, each of thefirst and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K, wherein numbering is according to EUnumbering.

In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst or second monomer includes pI variants (including those shown inFIG. 4 ). In exemplary embodiments, the constant domain(CH1-hinge-CH2-CH3) of the first or second monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering.

In some embodiments, the 2+1 CLC format antibody provided herein furtherincludes FcRn variants M428L/N434S, wherein numbering is according to EUnumbering.

In exemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q; each ofthe first and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K; and the constant domain(CH1-hinge-CH2-CH3) of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering. In some embodiments, the 2+1 CLC format antibody providedherein further includes FcRn variants M428L/N434S, wherein numbering isaccording to EU numbering.

In some embodiments, the CH1-hinge-CH2-CH3 of the second monomercomprises amino acid variantsL368D/K370S/N208D/Q295E/N384D/Q418E/N421D/E233P/L234V/L235A/G236del/S267K,and the first Fc domain comprises amino acid variantsS364K/E357Q/E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering.

In some embodiments, each of the two first binding domains binds a tumorassociated antigen (TAA) and the second binding domain binds CD28 (seeFIG. 34D). Any suitable CD28 binding domain can be included in thesubject 2+1 CLC format antibody, including any of the CD28 bindingdomains provided herein. In some embodiments, the CD28 binding domain isone of the following CD28 binding domains or a variant thereof:1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71, 1A7[CD28]_H1.1_L1.71,1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0, TGN1412_H1L1,341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1(FIGS. 18-21 and 23 and Sequence Listing). In exemplary embodiments, theCD28 binding domain includes a 1A7[CD28]_H1.14 variable heavy domain. Insome embodiments, the CD28 binding domain includes a 1A7[CD28]_H1.14variable heavy domain or variant thereof and a light variable domain ofany of the CD28 binding domains provided herein. In exemplaryembodiments, the CD28 binding domain is 1A7[CD28]_H1.14L1 or a variantthereof.

In some embodiments of the mAb-scFv format, the anti-CD28 ABD has a VHdomain with an amino acid sequence selected from the group consisting ofSEQ ID NO: 870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ IDNO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597,SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ IDNO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611,SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ IDNO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198,SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ IDNO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637,SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ IDNO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651,SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ IDNO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQID NO:671 and SEQ ID NO:672, and a VL domain with an amino acid sequenceselected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657,SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ IDNO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671,SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ IDNO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685,SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ IDNO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699,SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ IDNO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713,SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ IDNO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727,SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ IDNO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741,SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ IDNO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755,SEQ ID NO:1200 and SEQ ID NO:756.

In some embodiments, each of the two first binding domains binds a tumorassociated antigen (TAA). In certain embodiments, the two first bindingdomains bind the same TAA. Suitable TAAs include any of the TAAsdisclosed herein. In exemplary embodiments, the TAA is B7H3. Anysuitable B7H3 binding domain can be included in subject 2+1 CLC formatantibody, including any of the B7H3 binding domains provided herein. Insome embodiments, the B7H3 binding domain is one of the following B7H3binding domains or a variant thereof: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, m1704 (FIGS. 26-31 and the SequenceListing).

In some embodiments, the anti-B7H3 ABD has a VL domain with an aminoacid sequence selected from the group consisting of a variable heavydomain with an amino acid sequence selected from the group consisting ofSEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ IDNO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508,SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ IDNO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523,SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ IDNO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537,SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ IDNO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551,SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ IDNO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565,SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ IDNO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579,SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583 and SEQ IDNO:584; and a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO:874 and SEQ ID NO: 932.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:946; and a variable light domain havingthe amino acid sequence of SEQ ID NO:950.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:956; and a variable light domain havingthe amino acid sequence of SEQ ID NO:960.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:964; and a variable light domain havingthe amino acid sequence of SEQ ID NO:968.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:972; and a variable light domain havingthe amino acid sequence of SEQ ID NO:976.

In some embodiments, the anti-B7H3 ABD has a VH domain and VL domainwith amino acid sequences selected from the pairs of a) SEQ ID NOs: 89and 93 from omburamab, b) SEQ ID NOs:97 and 101 from enoblituzumab, c)SEQ ID NOs:105 and 109 from BRCA84D, d) SEQ ID NOs:113 and 117 fromBRCA69D, e) SEQ ID NOs:121 and 125 from PRCA157, f) SEQ ID NOs:129 and133 from huPRCA157, g) SEQ ID NOs:137 and 141 from Mab-D; h) SEQ IDNOs:145 and 149 humAb-D; i) SEQ ID NOs:153 and 157 from m30; j) SEQ IDNOs:161 and 165 from M30-H1-L4, k) SEQ ID NOs:169 and 173 SP265; l) SEQID NOs:177 and 181 from S10-H50L58; m) SEQ ID NOs:185 and 189 from 8H9,n) SEQ ID NOs:193 and 197 from m852; o) SEQ ID NOs:201 and 205 fromm857; p) SEQ ID NOs:209 and 213 from m8524; q) SEQ ID NOs:217 and 221from 1-1; r) SEQ ID NOs:225 and 229 from 1-2; s) SEQ ID NOs:233 and 237from 1-4; t) SEQ ID NOs:241 and 245 from 1-5; u) SEQ ID NOs:249 and 253from 1-7; v) SEQ ID NOs:257 and 261 from 2-5; w) SEQ ID NOs:265 and 269from 2-8; x) SEQ ID NOs: 273 and 277 from chAb2; y) SEQ ID NOs:281 and285 chAb3; z) SEQ ID NOs:289 and 293 from chAb4; aa) SEQ ID NOs:297 and301 from chAb18; bb) SEQ ID NOs:305 and 309 from chAb13; cc) SEQ IDNOs:313 and 317 from chAb12; dd) SEQ ID NOs:321 and 325 from chAb14; ee)SEQ ID NOs:329 and 333 from chAb6; ff) SEQ ID NOs:337 and 341 fromchAb11, gg) SEQ ID NOs:345 and 349 from chAB16; hh) SEQ ID NOs:353 and357 from chAb10; ii) SEQ ID NOs:361 and 365 from ChAb7; jj) SEQ IDNOs:369 and 373 from chAb8, kk) SEQ ID NOs:377 and 381 from chAb17; ll)SEQ ID NOs:385 and 389 from chAb5, mm) SEQ ID NOs:393 and 397 fromhuAb3v2.5, nn) SEQ ID NOs:401 and 405 from huAb3v2.6, pp) SEQ ID NOs:409and 413 from huAb13v1, qq) SEQ ID NOs:417 and 421 from TPP-5706, rr) SEQID NOs:425 and 429 from TPP-6642; ss) SEQ ID NOs:433 and 437 fromTPP-6850, tt) SEQ ID NOs:441 and 445 from TPP-3803, uu) SEQ ID NOs:449and 453 from TRL4542, vv) SEQ ID NOs:457 and 461 from h1702, ww) SEQ IDNOs:465 and 469 from h1703, xx) SEQ ID NOs:473 and 477 from huA3, yy)SEQ ID NOs:481 and 485 from huA9 and zz) SEQ ID NOs: 489 and 493 fromm1704. See FIG. 17 from U.S. Ser. No. 63/092,272.

In exemplary embodiments, the B7H3 binding domain includes a1A7[CD28]_H1.14 variable heavy domain. In some embodiments, the B7H3binding domain includes a 2E4A3.189[B7H3]_H1.22 variable heavy domainand a light variable domain of any of the CD28 or B7H3 binding domainsprovided herein. In exemplary embodiments, the B7H3 binding domainincludes a 2E4A3.189[B7H3]_H1.22 variable heavy domain or a variantthereof and a 1A7[CD28]_L1 variable light domain or a variant thereof.

In some embodiments, the 2+1 CLC format antibody includes two firstbinding domains that each bind B7H3 and a second binding domain thatbinds CD28. In some embodiments, the variable heavy domain of each ofthe first binding domains (i.e., the B7H3 binding domains) is a2E4A3.189[B7H3]_H1.22 variable heavy domain or variant thereof. Inparticular embodiments, the variable heavy domain of the second bindingdomain (i.e., the CD28 binding domain) is a 1A7[CD28]_H1.14 variableheavy domain or variant thereof. In some embodiments, the 2+1 CLC formatantibody includes a common light chain that includes the variable lightdomain of any of the CD28 or B7H3 binding domains provided herein. Insome embodiments, the variable light domain is a 1A7[CD28]_L1 variablelight domain or a variant thereof. Exemplary anti-B7H3×anti-CD28bispecific antibodies in the 2+1 CLC format are depicted in FIG. 38 .

FIG. 13 depicts sequences for “CH1+ half hinge” domain linker that finduse in embodiments of the 2+1 CLC format. In the 2+1 CLC format, the“CH1+ half hinge” sequences find use linking the first variable heavydomain (V_(H)) to the second VH domain on the Fab-Fab-Fc side of thebispecific antibody.

In some embodiments, the second monomer comprises the amino acidsequence of SEQ ID NO:1019, the first monomer comprises the amino acidsequence of SEQ ID NO:1020, and the light chain has the amino acidsequence of SEQ ID NO:1021.

5. 2+1 mAb-scFv Format

One heterodimeric antibody format that finds particular use in thesubject bispecific antibodies provided herein (e.g., anti-CD28×anti-B7H3antibody) is the 2+1 mAb-scFv format shown in FIG. 33E. This antibodyformat includes three antigen binding domains: two Fab portions and anscFv that is attached to the C-terminal of one of the heavy chains. Insome embodiments of this format, the Fab portions each bind a tumorassociated antigen (TAA), in this case, human B7H3 and the “extra” scFvdomain binds CD28. That is, this mAb-scFv format is a trivalentantibody.

In these embodiments, the first chain or monomer comprises, from N- toC-terminal, VH1-CH1-hinge-CH2-CH3, the second monomer comprises, from N-to C-terminal, VH1-CH1-hinge-CH2-CH3-domain linker-scFv domain, wherethe scFv domain comprises a second VH (VH2), a second VL (VL2) and ascFv linker. As for all the scFv domains herein, the scFv domain can bein either orientation, from N- to C-terminal, VH2-scFv linker-VL2 orVL2-scFv linker-VH2. Accordingly, the second monomer may comprise, fromN- to C-terminal, VH1-CH1-hinge-CH2-CH3-domain linker-VH2-scFvlinker-VL2 or VH1-CH1-hinge-CH2-CH3-domain linker-VL2-scFv linker-VH2.The composition also comprises a light chain, VL1-CL. In theseembodiments, the VH1-VL1 each form a first ABD and the VH2-VL2 form asecond ABD. In some embodiments, the first ABD binds to a tumor targetantigen, including human B7H3, and the second ABD binds human CD28.

In some embodiments, the first and second Fc domains of the 2+1 mAb-scFvformat antibody are variant Fc domains that include heterodimerizationskew variants (e.g., a set of amino acid substitutions as shown in FIGS.3 and 9 ). Particularly useful heterodimerization skew variants includeS364K/E357Q:L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K;T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L; K370S: S364K/E357Q;T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C (EUnumbering)). In exemplary embodiments, one of the first or secondvariant Fc domains includes heterodimerization skew variants L368D/K370Sand the other of the first or second variant Fc domains includesheterodimerization skew variants S364K/E357Q, wherein numbering isaccording to EU numbering. In exemplary embodiments, the first variantFc domain includes heterodimerization skew variants L368D/K370S and thesecond variant Fc domain includes heterodimerization skew variantsS364K/E357Q, wherein numbering is according to EU numbering.

In some embodiments, the variant Fc domains include ablation variants(including those shown in FIG. 5 ). In some embodiments, each of thefirst and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K, wherein numbering is according to EUnumbering.

In some embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst monomer includes pI variants (including those shown in FIG. 4 ).In exemplary embodiments, the constant domain (CH1-hinge-CH2-CH3) of thefirst monomer includes pI variants N208D/Q295E/N384D/Q418E/N421D,wherein numbering is according to EU numbering.

In some embodiments, the scFv of the 2+1 mAb-scFv format antibodyprovided herein includes a charged scFv linker (including those shown inFIG. 6 ). In some embodiments, the 2+1 mAb-scFv format antibody providedherein includes FcRn variants M428L/N434S, wherein numbering isaccording to EU numbering.

In exemplary embodiments, the first variant Fc domain includesheterodimerization skew variants L368D/K370S and the second variant Fcdomain includes heterodimerization skew variants S364K/E357Q; each ofthe first and second variant Fc domains include ablation variantsE233P/L234V/L235A/G236_/S267K; and the constant domain(CH1-hinge-CH2-CH3) of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering. In some embodiments, the scFv of the 2+1 mAb-scFv formatantibody provided herein includes a (GKPGS)₄ charged scFv linker. Insome embodiments, 2+1 mAb-scFv format antibody provided herein includesFcRn variants M428L/N434S, wherein numbering is according to EUnumbering.

In some embodiments, the scFv of the second monomer of the 2+1Fab₂-scFv-Fc format antibody is a CD28 binding and the VH1 of the firstand second monomer and the VL1 of the common light chain each formbinding domains that bind a tumor associated antigen (TAA, e.g., B7H3)(see FIG. 26B). Any suitable CD28 binding domain can be included insubject 2+1 mAb-scFv format antibody, including any of the CD28 bindingdomains provided herein. In some embodiments, the CD28 binding domain isone of the following CD28 binding domains or a variant thereof:1A7[CD28]_H1L1, 1A7[CD28]_H1.14L1, 1A7[CD28]_H1_L1.71,1A7[CD28]_H1.1_L1.71, 1A7[CD28]_H1.14_L1.71, CD28.3[CD28]_H0L0,TGN1412_H1L1, 341VL34[CD28]_H1L1, 341VL36[CD28]_H1L1, 281VL4[CD28]_H1L1,HuTN228[CD28]_H1L1, PV1[CD28]_H0L0, m9.3[CD28]_H0L0, hu9.3[CD28]_H1L1(FIGS. 18-21 and 23 and Sequence Listing).

In some embodiments of the mAb-scFv format, the anti-CD28 ABD has a VHdomain with an amino acid sequence selected from the group consisting ofSEQ ID NO: 870, SEQ ID NO:585, SEQ ID NO:586, SEQ ID NO:587, SEQ IDNO:588, SEQ ID NO:589, SEQ ID NO:590, SEQ ID NO:591, SEQ ID NO:592, SEQID NO:593, SEQ ID NO:594, SEQ ID NO:595, SEQ ID NO:596, SEQ ID NO:597,SEQ ID NO:598, SEQ ID NO:599, SEQ ID NO:600, SEQ ID NO:601, SEQ IDNO:602, SEQ ID NO:603, SEQ ID NO:604, SEQ ID NO:605, SEQ ID NO:606, SEQID NO:607, SEQ ID NO:608, SEQ ID NO:609, SEQ ID NO:610, SEQ ID NO:611,SEQ ID NO:612, SEQ ID NO:613, SEQ ID NO:614, SEQ ID NO:615, SEQ IDNO:616, SEQ ID NO:617, SEQ ID NO:618, SEQ ID NO:619, SEQ ID NO:620, SEQID NO:621, SEQ ID NO:622, SEQ ID NO:623, SEQ ID NO:624, SEQ ID NO:1198,SEQ ID NO:1199, SEQ ID NO:625, SEQ ID NO:626, SEQ ID NO:627, SEQ IDNO:628, SEQ ID NO:629, SEQ ID NO:630, SEQ ID NO:631, SEQ ID NO:632, SEQID NO:633, SEQ ID NO:634, SEQ ID NO:635, SEQ ID NO:636, SEQ ID NO:637,SEQ ID NO:638, SEQ ID NO:639, SEQ ID NO:640, SEQ ID NO:641, SEQ IDNO:642, SEQ ID NO:643, SEQ ID NO:644, SEQ ID NO:645, SEQ ID NO:646, SEQID NO:647, SEQ ID NO:648, SEQ ID NO:649, SEQ ID NO:650, SEQ ID NO:651,SEQ ID NO:652, SEQ ID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ IDNO:656, SEQ ID NO:657, SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:670, SEQID NO:671 and SEQ ID NO:672, and a VL domain with an amino acid sequenceselected from the group consisting of SEQ ID NO:874, SEQ ID NO:652, SEQID NO:653, SEQ ID NO:654, SEQ ID NO:655, SEQ ID NO:656, SEQ ID NO:657,SEQ ID NO:658, SEQ ID NO:659, SEQ ID NO:660, SEQ ID NO:661, SEQ IDNO:662, SEQ ID NO:663, SEQ ID NO:664, SEQ ID NO:665, SEQ ID NO:666, SEQID NO:667, SEQ ID NO:668, SEQ ID NO:669, SEQ ID NO:670, SEQ ID NO:671,SEQ ID NO:672, SEQ ID NO:673, SEQ ID NO:674, SEQ ID NO:675, SEQ IDNO:676, SEQ ID NO:677, SEQ ID NO:678, SEQ ID NO:679, SEQ ID NO:680, SEQID NO:681, SEQ ID NO:682, SEQ ID NO:683, SEQ ID NO:684, SEQ ID NO:685,SEQ ID NO:686, SEQ ID NO:687, SEQ ID NO:688, SEQ ID NO:689, SEQ IDNO:690, SEQ ID NO:691, SEQ ID NO:692, SEQ ID NO:693, SEQ ID NO:694, SEQID NO:695, SEQ ID NO:696, SEQ ID NO:697, SEQ ID NO:698, SEQ ID NO:699,SEQ ID NO:700, SEQ ID NO:701, SEQ ID NO:702, SEQ ID NO:703, SEQ IDNO:704, SEQ ID NO:705, SEQ ID NO:706, SEQ ID NO:707, SEQ ID NO:708, SEQID NO:709, SEQ ID NO:710, SEQ ID NO:711, SEQ ID NO:712, SEQ ID NO:713,SEQ ID NO:714, SEQ ID NO:715, SEQ ID NO:716, SEQ ID NO:717, SEQ IDNO:718, SEQ ID NO:719, SEQ ID NO:720, SEQ ID NO:721, SEQ ID NO:722, SEQID NO:723, SEQ ID NO:724, SEQ ID NO:725, SEQ ID NO:726, SEQ ID NO:727,SEQ ID NO:728, SEQ ID NO:729, SEQ ID NO:730, SEQ ID NO:731, SEQ IDNO:732, SEQ ID NO:733, SEQ ID NO:734, SEQ ID NO:735, SEQ ID NO:736, SEQID NO:737, SEQ ID NO:738, SEQ ID NO:739, SEQ ID NO:740, SEQ ID NO:741,SEQ ID NO:742, SEQ ID NO:743, SEQ ID NO:744, SEQ ID NO:745, SEQ IDNO:746, SEQ ID NO:747, SEQ ID NO:748, SEQ ID NO:749, SEQ ID NO:750, SEQID NO:751, SEQ ID NO:752, SEQ ID NO:753, SEQ ID NO:754, SEQ ID NO:755,SEQ ID NO:1200 and SEQ ID NO:756.

In some embodiments, the VH1 of the first and second monomer and the VL1of the common light chain of the 2+1 Fab₂-scFv-Fc format antibody eachform a binding domain that binds a tumor associated antigen (TAA) (seeFIG. 26B). Suitable TAAs include any of the TAAs disclosed herein. Inexemplary embodiments, the TAA is B7H3. Any suitable B7H3 binding domaincan be included in subject 2+1 Fab₂-scFv-Fc format antibody, includingany of the B7H3 binding domains provided herein. In some embodiments,the B7H3 binding domain is one of the following B7H3 binding domains ora variant thereof: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, omburtamab,enoblituzumab, BRCA84D, BRCA69D, PRCA157, huPRCA157, mAb-D, humAb-D,M30, M30-H1-L4, SP265, S10-H50L58, 8H9, m852, m857, m8524, 1-1, 1-2,1-4, 1-5, 1-7, 2-5, 2-8, chAb2, chAb3, chAb4, chAb18, chAb13, chAb12,chAb14, chAb6, chAb11, chAb16, chAb10, chAb7, chAb8, chAb17, chAb5,huAb3v2.5, huAb3v2.6, huAb13v1, TPP-5706, TPP-6642, TPP-6850, TPP-3803,TRL4542, h1702, h1703, huA3, huA9, m1704 (FIGS. 26-31 and the SequenceListing).

In some embodiments, the anti-B7H3 ABD has a VL domain with an aminoacid sequence selected from the group consisting of a variable heavydomain with an amino acid sequence selected from the group consisting ofSEQ ID NO:518, SEQ ID NO:928, SEQ ID NO:497, SEQ ID NO:498, SEQ IDNO:499, SEQ ID NO:500, SEQ ID NO:501, SEQ ID NO:502, SEQ ID NO:503, SEQID NO:504, SEQ ID NO:505, SEQ ID NO:506, SEQ ID NO:507, SEQ ID NO:508,SEQ ID NO:509, SEQ ID NO:510, SEQ ID NO:511, SEQ ID NO:512, SEQ IDNO:513, SEQ ID NO:514, SEQ ID NO:515, SEQ ID NO:516, SEQ ID NO:517, SEQID NO:519, SEQ ID NO:520, SEQ ID NO:521, SEQ ID NO:522, SEQ ID NO:523,SEQ ID NO:524, SEQ ID NO:525, SEQ ID NO:526, SEQ ID NO:527, SEQ IDNO:528, SEQ ID NO:529, SEQ ID NO:530, SEQ ID NO:531, SEQ ID NO:532, SEQID NO:533, SEQ ID NO:534, SEQ ID NO:535, SEQ ID NO:536, SEQ ID NO:537,SEQ ID NO:538, SEQ ID NO:539, SEQ ID NO:540, SEQ ID NO:541, SEQ IDNO:542, SEQ ID NO:543, SEQ ID NO:544, SEQ ID NO:545, SEQ ID NO:546, SEQID NO:547, SEQ ID NO:548, SEQ ID NO:549, SEQ ID NO:550, SEQ ID NO:551,SEQ ID NO:552, SEQ ID NO:553, SEQ ID NO:554, SEQ ID NO:555, SEQ IDNO:556, SEQ ID NO:557, SEQ ID NO:558, SEQ ID NO:559, SEQ ID NO:560, SEQID NO:561, SEQ ID NO:562, SEQ ID NO:563, SEQ ID NO:564, SEQ ID NO:565,SEQ ID NO:566, SEQ ID NO:567, SEQ ID NO:568, SEQ ID NO:569, SEQ IDNO:570, SEQ ID NO:571, SEQ ID NO:572, SEQ ID NO:573, SEQ ID NO:574, SEQID NO:575, SEQ ID NO:576, SEQ ID NO:577, SEQ ID NO:578, SEQ ID NO:579,SEQ ID NO:580, SEQ ID NO:581, SEQ ID NO:582, SEQ ID NO:583 and SEQ IDNO:584; and a VL domain having the amino acid sequence selected from thegroup consisting of SEQ ID NO:874 and SEQ ID NO: 932.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:946; and a variable light domain havingthe amino acid sequence of SEQ ID NO:950.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:956; and a variable light domain havingthe amino acid sequence of SEQ ID NO:960.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:964; and a variable light domain havingthe amino acid sequence of SEQ ID NO:968.

In some embodiments, the anti-B7H3 ABD comprises a VH domain having theamino acid sequence of SEQ ID NO:972; and a variable light domain havingthe amino acid sequence of SEQ ID NO:976.

FIGS. 10-11 show some exemplary Fc domain sequences that are useful withthe 2+1 mAb-scFv format. The “monomer 1” sequences depicted in FIG. 10typically refer to the Fc domain of the “Fab-Fc heavy chain” and the“monomer 2” sequences refer to the Fc domain of the “Fab-Fc-scFv” heavychain.” In addition, FIGS. 12-14 provides exemplary CH1 (optionallyincluding hinge or half-hinge domains) that can be used in either the“Fab-Fc heavy chain” monomer or the “Fab-Fc-scFv” heavy chain.” FIG. 15provides exemplary hinge domains that may be used in either the “Fab-Fcheavy chain” monomer or the “Fab-Fc-scFv” heavy chain.” Further, FIG. 16provides useful CL sequences that can be used with this format.

6. Monospecific, Monoclonal Antibodies

As will be appreciated by those in the art, the novel Fv sequencesoutlined herein can also be used in both monospecific antibodies (e.g.,“traditional monoclonal antibodies”) or non-heterodimeric bispecificformats. Accordingly, the present invention provides monoclonal(monospecific) antibodies comprising the 6 CDRs and/or the vh and vlsequences from the figures, generally with IgG1, IgG2, IgG3 or IgG4constant regions, with IgG1, IgG2 and IgG4 (including IgG4 constantregions comprising a S228P amino acid substitution) finding particularuse in some embodiments. That is, any sequence herein with a “H_L”designation can be linked to the constant region of a human IgG1antibody.

In some embodiments, the monospecific antibody is a B7H3 monospecificantibody. In certain embodiments, the monospecific anti-B7H3 antibodyincludes the 6 CDRs of any of the following B7H3 antigen bindingdomains: 2E4A3.189[B7H3]_H1L1, 2E4A3.189[B7H3]_H1/1A7[CD28]_L1,2E4A3.189[B7H3]_H1.22_L1, 2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1,6A1[B7H3]_H1L1, 3C4[B7H3]_H1L1.1, and 4F12[B7H3]_H2L1.1 (FIGS. 26-31 ).In some embodiments, the monospecific B7H3 antibody includes thevariable heavy domain and variable light domain of any of the followingB7H3 antigen binding domains: 2E4A3.189[B7H3]_H1L1,2E4A3.189[B7H3]_H1/1A7[CD28]_L1, 2E4A3.189[B7H3]_H1.22_L1,2E4A3.189[B7H3]_H1.22/1A7[CD28]_L1, 6A1[B7H3]_H1L1, 3C4[B7H3]_H1L1.1,and 4F12[B7H3]_H2L1.1 (FIGS. 26-31 ).

In some embodiments, the monospecific antibody is a CD28 monospecificantibody. In certain embodiments, the monospecific anti-CD28 antibodyincludes the 6 CDRs of any of the following CD28 antigen bindingdomains: 1A7[CD28]_H1L1, and 1A7[CD28]_H1.14_L1; (FIGS. 18 and 19 ). Insome embodiments, the monospecific anti-CD28 antibody includes thevariable heavy domain and variable light domain of any of the CD28antigen binding domains: 1A7[CD28]_H1L1, and 1A7[CD28]_H1.14_L1 (FIGS.18 and 19 ).

VI. Nucleic Acids

In another aspect, provided herein are nucleic acid compositionsencoding the antigen binding domains and anti-B7H3 and anti-CD28antibodies provided herein (e.g., αB7H3ΔαCD28 bispecific antibodies).

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format and scaffold of the heterodimericprotein. Thus, for example, when the format requires three amino acidsequences, such as for the 1+1 Fab-scFv-Fc or 2+1 Fab₂-scFv-Fc formats,1+1 CLC and 2+1 CLC formats, three polynucleotides can be incorporatedinto one or more expression vectors for expression. In exemplaryembodiments, each polynucleotide is incorporated into a differentexpression vector.

As is known in the art, the nucleic acids encoding the components of thebinding domains and antibodies disclosed herein can be incorporated intoexpression vectors as is known in the art, and depending on the hostcells used to produce the heterodimeric antibodies of the invention.Generally the nucleic acids are operably linked to any number ofregulatory elements (promoters, origin of replication, selectablemarkers, ribosomal binding sites, inducers, etc.). The expressionvectors can be extra-chromosomal or integrating vectors.

The polynucleotides and/or expression vectors of the invention are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g., CHO cells), finding use inmany embodiments.

In some embodiments, polynucleotides encoding each monomer are eachcontained within a single expression vector, generally under differentor the same promoter controls. In embodiments of particular use in thepresent invention, each of these polynucleotides are contained ondifferent expression vectors. As shown herein and in U.S. 62/025,931,hereby incorporated by reference, different vector ratios can be used todrive heterodimer formation. That is, surprisingly, while the proteinscomprise first monomer: second monomer:light chains (in the case of manyof the embodiments herein that have three polypeptides comprising theheterodimeric antibody) in a 1:1:2 ratio, these are not the ratios thatgive the best results.

The antibodies and ABDs provided herein are made by culturing host cellscomprising the expression vector(s) as is well known in the art. Onceproduced, traditional antibody purification steps are done, including anion exchange chromatography step. As discussed herein, having the pIs ofthe two monomers differ by at least 0.5 can allow separation by ionexchange chromatography or isoelectric focusing, or other methodssensitive to isoelectric point. That is, the inclusion of pIsubstitutions that alter the isoelectric point (pI) of each monomer sothat such that each monomer has a different pI and the heterodimer alsohas a distinct pI, thus facilitating isoelectric purification of the“1+1 Fab-scFv-Fc” heterodimer (e.g., anionic exchange columns, cationicexchange columns). These substitutions also aid in the determination andmonitoring of any contaminating dual scFv-Fc and mAb homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

VII. Biological and Biochemical Functionality of the Anti-CD28×Anti-TAAAntibodies

Generally the bispecific anti-CD28×anti-TAA antibodies described herein(e.g., anti-CD28×anti-B7H3) are administered to patients with cancer(e.g., a B7H3 associated cancer), and efficacy is assessed, in a numberof ways as described herein. Thus, while standard assays of efficacy canbe run, such as cancer load, size of tumor, evaluation of presence orextent of metastasis, etc., immuno-oncology treatments can be assessedon the basis of immune status evaluations as well. This can be done in anumber of ways, including both in vitro and in vivo assays.

A. Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

VIII. Treatments

Once made, the compositions of the invention find use in a number ofoncology applications, by treating cancer, generally by enhancing immuneresponses (e.g., T cell activation and proliferation), particularly whenused with anti-cancer therapies such as anti-PD1 and anti-tumorbispecific antibodies. In some embodiments, the antibodies providedherein enhance immune responses (e.g., T cell activation andproliferation) by providing agonistic co-stimulation of T cells in themicroenvironment of tumors expressing a TAA of interest (e.g., B7H3).

In some embodiments, the anti-CD28×anti-TAA bispecific antibodiesprovided herein are administered with an anti-tumor therapy including,for example, a checkpoint inhibitor (e.g., anti-PD1 antibody) oranti-tumor bispecific antibodies.

A. Anti-CD28×Anti-TAA/Anti-TAA Bispecific Antibody

In some embodiments, the anti-CD28×anti-TAA bispecific antibodiesprovided herein are administered with an anti-tumor bispecific antibodythat is a T-cell engaging bispecific antibody, such as those that bindto human Cd3.

In classic T cell/APC interaction, there is a first signal provided byTCR reactivity with peptide-MHC (Signal 1) and a second signal providedby CD28 crosslinking by CD80/CD86 being expressed on APCs (Signal 2)which together fully activate T cells (see FIG. 31A). In contrast, onlythe first signal is provided in treatment with CD3 bispecific antibodiesthat target a TAA (i.e., anti-CD3×anti-TAA bispecific antibodies).

Without being bound by any particular theory of operation, it isbelieved that the anti-CD28×anti-TAA bispecific antibodies providedherein can enhance the anti-tumor response of an anti-CD3×anti-TAAbispecific antibody by CD28 costimulation (see FIG. 31B and Examples 4Eand 4F). Thus, in one aspect, provided herein are methods of methods oftreating a cancer in a patient by administering the patient ananti-CD3×anti-TAA bispecific antibody and an anti-CD28×anti-TAAbispecific antibody provided herein. In some cases, the TTA is the samein both antibodies; thus, for example, there can be co-administration ofan anti-CD28 X B7H3 bispecific antibody with an anti-CD3 X B7H3antibody. In some cases, the TTAs are different. In some embodiments,the administration of the anti-CD3×anti-TAA bispecific antibody andanti-CD28×anti-TAA bispecific antibody enhances an immune responseagainst the tumor in the patient. In some embodiments, theanti-CD3×anti-TAA bispecific antibody and anti-CD28×anti-TAA binds todifferent TAAs on the same tumor. In exemplary embodiments, theanti-CD28×anti-TAA is an anti-CD28×anti-B7H3 antibody provided herein.

B. Anti-CD28×Anti-TTA/Checkpoint Inhibitor

In some embodiments, the anti-CD28×anti-TAA bispecific antibodiesprovided herein are administered with a checkpoint inhibitor (e.g.,anti-PD1 antibody). Without being bound by any particular theory ofoperation, it is believed that checkpoint blockade (e.g. PD-1 blockade)is a useful therapeutic modality to stack with engagement of T cellcostimulatory receptors on TILs with agonistic anti-CD28×anti-TAAbispecific antibodies as it would provide broad utility in solid tumorsand circumvent CTLA4 inhibition of the CD28 pathway. Thus, in anotheraspect provided herein is a method of treating a cancer in a patient byadministering the patient an anti-CD28×anti-TAA bispecific antibodyprovided herein and a checkpoint inhibitor. In some embodiments, theadministration of the anti-CD28×anti-TAA bispecific antibody andcheckpoint inhibitor enhances an immune response against the tumor inthe patient. In some embodiments, the checkpoint inhibitor is a PD-1,PD-L1, or CTLA4 inhibitor. In exemplary embodiments, the PD-1 inhibitoris an anti-PD-1, anti-PD-L1 or anti-CTLA4 antibody.

C. Administrative Modalities

The antibodies provided herein administered to a subject, in accord withknown methods, such as intravenous administration as a bolus or bycontinuous infusion over a period of time.

D. Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition.

By “positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the bispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in USPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein.

Background

While checkpoint blockade immunotherapies have proven to be effective,many patients nonetheless fail to achieve a response. Engagement of Tcell costimulatory receptors on TILs with agonistic antibodies couldprovide an additional positive signal capable of overcoming negativesignals of immune checkpoints and may be a useful therapeutic modalityto stack with checkpoint blockade. However, systemic agonism ofcostimulatory receptors may nonetheless result in systemic toxicity.B7H3 has been found to be broadly overexpressed in cancer cells andtumor vascular cells and may be useful as a tumor target. Accordingly,αB7H3ΔαCD28 bispecific antibodies (bsAbs) were engineered with the aimto target agonistic CD28 binding domains to the tumor environmentthereby reducing the potential for systemic toxicity.

Example 1: CD28 Binding Domains

1A: Novel CD28 Binding Domains

An approach considered to avoid the superagonism associated with TGN1412was to generate novel CD28 binding domains having lower affinity bindingto CD28 and/or binding to a different CD28 epitope than TGN1412. In onecampaign to generate such novel CD28 binding domains, in-house de novophage libraries were panned against CD28. In another campaign, rathybridomas were used to generate additional CD28 binding domains.

1A(a): Phage-Derived Clone 1A7

It should be noted that this phage library utilized a human germline VLwith diversity introduced into the LCDR3. The amino acid sequences forexemplary phage-derived clone 1A7 are depicted in FIG. 18 .

The phage-derived clones were formatted as bivalent mAbs to investigatetheir binding characteristics. Plasmids containing the variable heavyand variable light domains of select clones were constructed by Gibsonassembly and subcloned into a pTT5 expression vector containing thecoding sequence for the IgG1 constant regions (withE233P/L234V/L235A/G236del/S67K ablation variants). DNA was transfectedin HEK293E for expression and resulting bivalent mAbs were purified fromthe supernatant using protein A chromatography.

Affinity of the phage-derived bivalent mAbs for CD28 was screened usingOctet, a BioLayer Interferometry (BLI)-based method. Experimental stepsfor Octet generally include the following: Immobilization (capture ofligand to a biosensor); Association (dipping of ligand-coated biosensorsinto wells containing the analyte); and Dissociation (returning ofbiosensors to well containing buffer). The resulting apparentdissociation constant (KD_(app)) are depicted in FIG. 24 for XENP28428(based on clone 1A7) and additional phage-derived comparators.

Binding of the phage-derived bivalent mAbs to cell-surface CD28 wasinvestigated. Human PBMCs were incubated with indicated concentrationsof XENP28428 or comparator phage-derived mAbs for 1 hour at 4° C. Cellswere then then stained with Alexa Fluor® 647 AffiniPure F(ab′)₂ FragmentGoat Anti-Human IgG, Fcγ fragment specific secondary antibody (JacksonImmunoResearch, West Grove, Pa.) for 1 hour at 4° C. and analyzed byflow cytometry. The data (FIG. 25 ) show that the phage-derived mAbswere able to bind human PBMCs, although with much weaker maximum bindingthan prior art anti-CD28 mAb HuTN228 (XENP27181, sequences for which aredepicted in FIG. 23 ).

In view of the weaker CD28 binding, 1A7 was further affinity engineeredby introducing substitutions into the VH and/or V_(L). Sequences forsuch affinity engineered VH and VL regions are depicted as SEQ ID NOS:585-756 (with illustrative sequences depicted in FIGS. 19-20 ); andsequences for illustrative affinity engineered V_(H)/VL pairs aredepicted in FIG. 21 . Consensus sequences for the FR and CDRs aredepicted in FIG. 44 . Affinity for illustrative affinity engineered 1A7V_(H)/VL pairs for CD28 are depicted in FIG. 22 . Notably, theorientation of the VH and VL domains in the context of an scFv affectsthe binding affinity. Additionally, formatting the VH and VL domains inthe context of a Fab domain (for use in common light chain bispecificmAb formats) as opposed to scFv also affects the binding affinity.

1B: Additional CD28 Binding Domains

V_(H), V_(L), and CDR sequences for additional CD28 binding domainswhich may find use in the αB7H3ΔαCD28 bsAbs of the invention aredepicted as SEQ ID NOs: 1-88.

Example 2: B7H3 Binding Domains

2A: Novel B7H3 Binding Domain

In one campaign to generate novel B7H3 binding domains, in-house de novophage libraries were panned against B7H3. In another campaign, rathybridomas were used to generate additional B7H3 binding domains.

2A(a): Phage-Derived Clone 2E4A3.189

It should be noted that this phage library was intended to discoverbinding domains suitable for use in common light chain bispecificantibody formats. Accordingly, it utilized the same human germline VL asin Example 1A(a) except without any diversity. The amino acid sequencesfor exemplary phage-derived clone 2E4A3.189 are depicted in FIG. 26 .While this phage-derived clone is useful for enabling common light chainbispecific antibody formats, it had very weak binding affinity for B7H3and required affinity engineering. As will be further described inExample 3B, the VH of 2E4A3.189 pairs productively with the VL of 1A7,but the VH of 1A7 does not pair productively with the VL of 2E4A3.189(despite one amino acid difference). Accordingly to improve affinity,2E4A3.189 was engineered with substitutions into the VH only, sequencesfor which are depicted as SEQ ID NOS: 497-584 and in FIG. 27 , andpaired with the VL of 1A7. Consensus sequences for the FR and CDRs aredepicted in FIG. 75 .

2B: Hybridoma-Derived Clones

B7H3 binding domains were obtained from rat and rabbit hybridoma andhumanized using string content optimization (see, e.g., U.S. Pat. No.7,657,380, issued Feb. 2, 2010). The amino acid sequences for exemplaryhumanized rat hybridoma-derived clones 6A1 and 3C4 and humanized rabbithybridoma-derived clones 4F12 and 38E2 are depicted respectively inFIGS. 28-31 . Binding affinities of the hybridoma clones (andaffinity-engineered 2E4A3.189 phage clone) for human and cynomolgus B7H3were determined in the context of 1+1 bsAb format (to obtain monovalentbinding affinities), data for which are depicted in FIG. 32 .

2C: Additional B7H3 Binding Domains

V_(H), V_(L), and CDR sequences for additional B7H3 binding domainswhich may find use in the B7H3×CD28 bsAbs of the invention are depictedas SEQ ID NOs: 89-96.

Example 3: Engineering αB7H3ΔαCD28 bsAbs

A number of formats for B7H3×CD28 bsAbs were conceived, illustrativeformats for which are outlined below and in FIG. 33 . It should be notedthat in each case, the CD28 bispecific antibodies are monovalent forCD28 and incorporate Fc variants to engineered to ablate FcγR binding(such as those depicted in FIG. 5 ) to avoid potential superagonism.

3A: Fab-scFv-Fc Formats

3A(a): 1+1 Fab-scFv-Fc Format

One format utilizing Fab domains and scFv is the 1+1 Fab-scFv-Fc format(depicted schematically in FIG. 34A) which comprises a first monomercomprising a single-chain Fv (“scFv”) with a first antigen bindingspecificity covalently attached to a first heterodimeric Fc domain, asecond monomer comprising a heavy chain variable region (V_(H))covalently attached to a complementary second heterodimeric Fc domain,and a light chain (LC) transfected separately so that a Fab domainhaving a second antigen binding specificity is formed with the variableheavy domain. Sequences for illustrative αB7H3ΔαCD28 bsAbs (based onbinding domains as described in Examples 1 and 2) in the 1+1 Fab-scFv-Fcformat are depicted in FIG. 35 .

3A(b): 2+1 Fab₂-scFv-Fc Format

Another such format is the 2+1 Fab₂-scFv-Fc format (depictedschematically in FIG. 34B) which comprises a first monomer comprising aVH domain covalently attached to an scFv (having a first antigen bindingspecificity) covalently attached to a first heterodimeric Fc domain, asecond monomer comprising a VH domain covalently attached to acomplementary second heterodimeric Fc domain, and a LC transfectedseparately so that Fab domains having a second antigen bindingspecificity are formed with the VH domains. Sequences for illustrativeαB7H3ΔαCD28 bsAbs (based on binding domains as described in Examples 1and 2) in the 2+1 Fab₂-scFv-Fc format are depicted in FIG. 36 .

3B: Common Light Chain Format

As described above in Examples 1 and 2, the phage library fordiscovering CD28 and B7H3 binding domains utilized the same humangermline V_(L), although the CD28 library included diversity in theLCDR3. It was found that the variable light domain of clone 1A7 differedfrom the variable light domain of anti-B7H3 clone 2E4A3.189 by only asingle amino acid in the LCDR3. Accordingly, the possible use of clone1A7 and clone 2E4A3.189 in a Common Light Chain construct wasconsidered. However, it was surprisingly found that the VH of 2E4A3.189paired productively with the VL of 1A7, but the VH of 1A7 did not pairproductively with the VL of 2E4A3.189 despite only having one amino aciddifference in the LCDR3. Further, as noted above, the phage-derivedclone 1A7 demonstrated much weaker binding than prior art anti-CD28 mAbHuTN228 providing an opportunity for affinity-optimization. Accordingly,affinity-optimization libraries were generated with focus first onsubstitutions only in the variable heavy domains of 1A7 and 2E4A3.189.The amino acid sequences for exemplary affinity-optimized 1A7 variabledomains H1.1 and H1.14 and affinity-optimized 2E4A3.189 variable heavydomain H1.3 and H1.22 are depicted respectively in FIGS. 19 and 27 .

3B(a): 1+1 Common Light Chain Format

One common light chain format is the 1+1 Common Light Chain (CLC) format(depicted schematically in FIG. 34C) which comprises a first monomercomprising VH1-CH1-hinge-CH2-CH3, a second monomer comprisingVH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VLpairs with the VH1 to form a binding domain with a first antigen bindingspecificity; and the VL pairs with the VH2 to form a binding domain witha second antigen binding specificity. Sequences for illustrativeαB7H3ΔαCD28 bsAbs (based on binding domains as described here) in the1+1 CLC format are depicted in FIG. 37 .

3B(b): 2+1 Common Light Chain Format

Another common light chain format is the 2+1 CLC format (depictedschematically in FIG. 34D) which comprises a first monomer comprisingVH1-CH1-hinge-VH1-CH1-hinge-CH2-CH3, a second monomer comprisingVH2-CH1-hinge-CH2-CH3, and a third monomer comprising VL-CL. The VLpairs with the first and second VH1 to form binding domains with a firstantigen binding specificity; and the VL pairs with the VH2 to form abinding domain with a second antigen binding specificity. Sequences forillustrative αB7H3ΔαCD28 bsAbs (based on binding domains as describedhere) in the 2+1 CLC format are depicted in FIG. 38 .

3C: 2+1 mAb-scFv Format

An additional format utilizing Fab domains and scFv is the 2+1 mAb-scFvformat (depicted schematically in FIG. 34E) which comprises a firstmonomer comprising a VH domain covalently attached to a firstheterodimeric Fc domain covalently attached to an scFv (having a firstantigen binding specificity), a second monomer comprising a VH domaincovalently attached to a complementary second heterodimeric Fc domain,and a LC transfected separately so that Fab domains having a secondantigen specificity are formed with the VH domains. Sequences forillustrative αB7H3ΔαCD28 bsAbs (based on binding domains as describedhere) in the 2+1 mAb-scFv format are depicted in FIG. 39 .

Example 4: Developing B7H3×CD28 bsAbs

In classic T cell/APC interaction, there is a first signal provided byTCR reactivity with peptide-MHC (Signal 1) and a second signal providedby CD28 crosslinking by CD80/CD86 being expressed on APCs (Signal 2)which together fully activate T cells (see FIG. 40A). In contrast intreatment with CD3 bispecifics, only the first signal is provided. Insome settings such as treatment of solid tumors, it might be useful tobuild in the CD28 signal which may be provided by a CD28 bispecific withthe idea to promote activation and proliferation through CD28costimulation (see FIG. 40B). Alternatively, where Signal 1 is alreadyprovided by endogenous TCR reactivity with neoepitopes, providing justSignal 2 with a CD28 bispecific antibody may be sufficient to enhanceanti-tumor activity. It may nonetheless be useful to stack the CD28signal with checkpoint blockade to mitigate any checkpoint mediatedrepression of the added CD28 signal (FIG. 41 ). The following sectionscharacterize B7H3×CD28 bispecific antibodies of the invention in thecontext of the foregoing. In this section, B7H3×CD28 bsAbs wereengineered in various formats and with various binding domains with anaim to optimize therapeutic properties.

4A: Tuning B7H3×CD28 bsAb Activity

The activity of 1+1 CD28 bispecific formats having monovalent binding tothe tumor-associated antigen was compared against the activity of 2+1CD28 bispecific formats having bivalent binding to the tumor-associatedantigen. 50,000 CD3+ T cells were incubated with A549 or SKOV-3 cancercells as a 10:1 effector:target ratio and treated with a dose titrationof the indicated B7H3×CD28 antibodies and plate bound 1 μg/mLplate-bound CD3 antibody (OKT3). 1 day post T cell seeding, cytokineswere measured using MSD assay (Meso Scale Discovery, Rockville, Md.).The data depicted in FIG. 42 show that both the B7H3×CD28 bispecificantibodies induced cytokine release by the T cells. Notably, XENP34339having bivalent B7H3 binding induced cytokine release more potently thanXENP34717 having monovalent B7H3 binding. It should be noted that thedifference in potency is less pronounced when using B7H3 binding domainshaving higher affinity binding (data not shown).

In another experiment, the impact of CD28 binding affinity on activitywas investigated. MCF7 cancer cell (transfected to express anti-CD3 scFvin order to provide the “Signal 1”) were incubated with effector cellsat a 1:1 effector:target ratio and the indicated concentrations ofXENP34339, XENP35612, XENP35611, and XENP34336. Each of the bsAbs werein the 2+1 CLC format. XENP34339, XENP35612, and XENP35611 each includedthe 2E4A3.189_H1.22_1A7_L1 B7H3 binding domain while XENP34336 includedthe lower affinity 2E4A3.189_H1.3_1A7_L1 B7H3 binding domain. XENP34339,XENP35612, XENP35611, and XENP34336 respectively included CD28 bindingdomains having 77 nM, 270 nM, 610 nM, and 440 nM binding affinity. Thedata as depicted in FIG. 43 show that increased affinity for CD28enhances potency of the B7H3×CD28 bsAb.

In another set of experiments, the activity of a panel of B7H3×CD28bsAbs in the presence of additional cancer cells was investigated. CD3+T cells were incubated with MDA-MB-2331, LnCAP, or DU145 cancer cells at1:1 E:T ratio, a constant dose of an illustrative B7H33×CD3 bsAb, anddose titration of B7H3×CD28 bsAbs. Data are depicted in FIG. 43 .Consistent with the above, increased affinity for CD28 enhances potencyof the B7H3×CD28 bsAb (XENP34398>XENP37808). Additionally, the data alsoindicate that increased affinity for B7H3 enhances potency of theB7H3×CD28 bsAb (e.g. XENP34398>XENP37810; XENP35151 andXENP35153>XENP34732; and XENP37807>XENP37982). Additionally, the dataindicate that the 2+1 CLC format is more potent in enhancing IL-2secretion in comparison to the 2+1 mAb-scFv format(XENP34398>XENP37807).

4B: Tuning CD28 bsAb Pharmacokinetic Profile

Next, the pharmacokinetic profile of various B7H3×CD28 bsAbs of theinvention were investigated.

In a first study, the pharmacokinetics of XENP34398 (having the 2+1 CLCformat), XENP36781 (having the 2+1 mAb-scFv format), and XENP34395(having the 2+1 central scFv format) were all tested in cynomolgus at arange of dosing levels. As depicted in FIG. 45 , XENP34398 in the 2+1CLC format was found to have significantly better pharmacokinetics thanthe 2+1 mAb-scFv format which was in turn slightly better than the 2+1Fab₂-scFv-Fc format, at each dose level tested. Although there wereother differences between these molecules in addition to the format(e.g. differences in the B7H3 binding domain), the data suggest the 2+1CLC format may be advantageous in the context of B7H3×CD28 bsAbs.

Additional B7H3×CD28 bsAbs were engineered with the various B7H3 bindingdomains (and it various formats) as described in Example 2 andpharmacokinetic profiles were investigated in another cynomolgus study.FIG. 46A-C depicts a comparison XENP34398, XENP37808 and XENP37810, eachof which are bsAbs in the 2+1 CLC format. XENP34398 and XENP37810 havethe same CD28 binding domain (1A7_H1.14_L1 Fab) but different affinityB7H3 binding domains (based on the same phage-derived clone, but thevariant in XENP34398 had higher affinity B7H3 binding than the variantin XENP37810). XENP34398 and XENP37808 have the same B7H3 binding domain(2E4A3.189_H1.22_1A7_L1) but different affinity CD28 binding domains(based on 1A7, but the variant in XENP34398 had tighter binding affinitythan the variant in XENP37808). FIG. 46D-F depicts a comparison ofXENP34732, XENP35151, and XENP35153, each of which are bsAbs in the 1+1Fab-scFv-Fc format and having the same CD28 binding domain (1A7_H1.14_L1scFv) but different B7H3 binding domains (respectively, 6A1, 4F12, and38E2). The data show that each of the 3 molecules having different B7H3binding domains had differing PK profiles despite being otherwiseidentical. In particular, XENP35151 (having 4F12 binding domain which asdescribed in Example 2B has much tighter binding affinity than either6A1 and 38E2) demonstrated worse PK profile in comparison to bothXENP34732 and XENP35153 (respectively having 6A1 and 38E2 bindingdomains). This suggests that at least in the 1+1 format, the bindingaffinity for B7H3 may impact pharmacokinetic profile. FIGS. 46G-Hdepicts comparison of XENP37807 and XENP37982, each of which are bsAbsin the 2+1 mAb-scFv format and having the same CD28 binding domain(1A7_H1.14_L1 scFv) but different B7H3 binding domains (respectively 2E4and 3C4).

4C: Summary of Select B7H3×CD28 bsAbs

FIG. 47 depicts a summary of properties of several of the B7H3×CD28bsAbs of the invention. It should be noted that some of the datadepicted in this summary table may not be the same experimental datadepicted elsewhere in the Working Examples as some of those illustrateexperimental data from earlier stages of development.

Example 5: Additional Characterization of Illustrative B7H3×CD28Bispecific Antibodies

Illustrative B7H3×CD28 bsAbs XENP34339 (or Xtend analog XENP34398) andXENP35612 (or Xtend analog XENP37808) were further characterized togenerally demonstrate useful properties of the B7H3×CD28 bispecificantibodies of the invention.

5A: XENP34339 Restores CD28 Signaling

CTLA-4 is an immune checkpoint receptor that competes with CD28 for CD28ligands CD80 and CD86; therefore, in the presence of CTLA-4 (as would befound in the tumor environment), CD28 signaling is dampened. Restorationof CD28 signaling by the CD28 bispecific antibodies of the inventionwere investigated in a mixed lymphocyte reaction. 100,000 CD3+ T cellswere incubated with 10,000 dendritic cells (STEMCELL Technologies,Vancouver, Canada) having high B7H3 expression and 1 μg/mL CTLA-4-Fcwere treated with a dose titration of B7H3×CD28 bispecific antibodyXENP34339. 3 days post T cell seeding, cytokines were measured using MSDassay. The data as depicted in FIG. 48 show that XENP34339 enablesendogenous CD28 signaling levels (i.e. absent introduced blockade byCTLA-4-Fc).

5B: XENP34339 Combines Productively with PD-1 Blockade

Checkpoint blockade (e.g. PD-1 blockade) may be a useful therapeuticmodality to stack with engagement of T cell costimulatory receptors onTILs with agonistic antibodies as it would provide broad utility insolid tumors and circumvent CTLA4 inhibition of CD28 pathway.Accordingly, the combination of B7H3×CD28 bispecific antibodiesXENP34339 and XENP34389 with XENP16432 (a bivalent anti-PD-1 mAb basedon the variable regions of nivolumab; sequences depicted in FIG. 17 )was investigated. 10,000 MDA-MB-231 cancer cells were treated with 100ng/ml HLA-A2*0201 restricted CMV pp65 (NLVPMVATV) peptide (NLV peptide)overnight. The following day, 100,000 CD3 enriched cells from a CMV+donor were added along with XENP16432 (PD-1 blockade; 10 μg/ml),XENP34339 (B7H3×CD28 in 2+1 CLC format with B7H3 binding domain based on2E4A3.189 and CD28 binding domain based on 1A7; 1 μg/ml), XENP34389(B7H3×CD28 in 2+1 Fab₂-scFv-Fc format with B7H3 binding domain based on6A1 and CD28 binding domain based on 1A7; 1 μg/ml), and combinations ofthe B7H3×CD28 with XENP16432. 1 day after treatment, cell supernatantwas assayed for cytokines using MSD assay (data for which are shown inFIG. 50 for experiments using CD3+ T cells from 2 different donors). Thedata show that incubation with XENP34339 alone induced cytokine releasefrom T cells and combined synergistically with PD-1 blockade to enhancecytokine release. Notably, XENP34389 (2+1 Fab₂-scFv-Fc format) did notcombine synergistically with PD-1 blockade. In a similar experiment,20,000 MCF7 cancer cells were seeded in the presence of 100 ng/mL NLVpeptide. After 24 hours, 200,000 CD3+ T cells (10:1 E:T) isolated from aCMV+PBMC donor and the test articles (PBS control, XENP34339 alone, PD-1mAb XENP16432 alone, or XENP34339+XENP16432) were added. After 6 days,cells were assessed via flow cytometry. Consistent with the above, thedata depicted in FIG. 51 PD-1 blockade enhances expansion ofNLV-tetramer positive CD8⁺ T cells by XENP34339.

To investigate whether the difference observed for XENP34339 resultedfrom the difference in B7H3 binding domain or the difference inbispecific antibody format, the component binding domains of XENP34339and XENP34389 were biophysically characterized using Octet. In a firstexperiment to determine the binding affinities of 2E4A3.189 and 6A1 forB7H3 antigen, XENP34339 and XENP34389 were reformatted to monovalentlybind to B7H3 antigen (respectively as XENP34717 and XENP34728, sequencesfor which are depicted in FIGS. 37 and 36 ). Anti-mouse Fc biosensorswere used to capture mouse Fc fusions of B7H3, either the fullextracellular V1C1-V2V2 domain or the individual V1C1 or V2C2 domains,and dipped into multiple concentrations of XENP34717 or XENP34728.Kinetic analyses were performed by global fitting of binding data with a1:1 Langmuir binding model. The resulting dissociation constant (K_(D))are depicted in FIG. 52 , and the data show that the 6A1 binding domainprovided slightly tighter binding to B7H3 than the 2E4A3.189. Next, thebinding affinities of the CD28 binding domains for CD28 antigen in the2+1 CLC format and the 2+1 Fab₂-scFv-Fc format was investigated.Anti-HIS capture (HIS1K) biosensors were used to capture CD28-Fc-Hisprotein and dipped into multiple concentrations of XENP34339 orXENP34389. Kinetic analyses were performed by global fitting of bindingdata with a 1:1 Langmuir binding model as well as steady state model.The resulting dissociation constant (KD) are depicted in FIG. 53 . Thedata show that the 2+1 CLC format enabled much tighter binding to CD28antigen than the 2+1 Fab₂-scFc-Fc format. Collectively, the datasuggests that the differences observed in the activity of XENP34339 andXENP34389 were due to the differences in bispecific antibody format.

5C: XENP34339 Overcomes Cancer Cell Resistance to CD3 Bispecifics at LowEffector to Target Ratios

It has been reported in literature that non-inflamed, cold tumors suchas prostate cancer have low effector:target ratio. Accordingly, cellkill at a 1:1 effector:target was assessed using xCELLigence Real TimeCell Analysis instrument (ACEA Biosciences, San Diego, Calif.). 2,500LNCaP cancer cells were first seeded. After 48 hours, freshly enrichedCD3+ T cells at an effector:target of 1:1 were added along withantibodies (αPSMA×αCD3 XENP31602 alone or XENP31602 in combination withXENP34339; sequences for XENP31602 are depicted in FIG. 54 ) at theindicated concentrations. Cell kill was recorded for 5 days post T cellseeding. The data as depicted in FIG. 55 show that XENP31602 alonestruggled to enhance cell kill in comparison to incubation of cancer andT cells alone indicating that there is a resistance to the CD3bispecific at the low 1:1 effector to target ratio.

Notably, addition of B7H3×CD28 overcomes cancer cell resistance to theCD3 bispecific. Although this experiment utilized a PSMA×CD3 bispecificantibody, it is reasonable to expect a similar outcome in combining theB7H3×CD28 bispecific antibodies of the invention with other CD3bispecific antibodies including those utilizing the CD3 binding domainsdepicted in FIG. 56 .

5D: XENP34339 Combines with PSMA×CD3 Bispecifics to Enhance ActivityOnly in the Presence of Both B7H3 and PSMA

10,000 cancer cells (LNCaP [PSMA+B7H3+], 22RV1 [PSMA+B7H3+], SKOV-3[PSMA−B7H3+], or OVCAR-8 [PSMA−B7H3+]) were first seeded. The followingday, freshly enriched CD3+ T cells were added at an effector:targetratio of 1:1 with 1 μg/ml XENP34339 in combination with a dose titrationof an illustrative CD3 bispecific (αPSMA×αCD3 XENP31602). One day post Tcell seeding, cytokines were measure using MSD assay and CD3+ T cellswere counted using flow cytometry, data for which are depicted in FIGS.57-60 . The data show that the CD3 bispecific XENP31602 alone inducedlittle to no T cell activity and proliferation at the low 1:1effector:target ratio. However in the presence of LNCaP and 22Rv1 whichare PSMA+B7H3+, the addition of αB7H3ΔαCD28 XENP34339 enhances theactivity of αPSMA×αCD3 XENP31602. Notably, however, in the presence ofSKOV-2 and OVCAR-8 which are PSMA−B7H3+, the addition of XENP343398 doesnot enhance activity. This requirement for both the tumor antigenassociated with the CD28 bispecific antibody and the tumor antigenassociated with the CD3 bispecific creates an AND gate useful forselectively targeting immune response to tumor cells which are morelikely to co-express multiple tumor-associated antigens. Thissynergistic AND gate may also enable activity on tumors having lowertarget densities wherein the tumor cells may express multipletumor-associated antigens albeit at low densities.

5E: Combining XENP34339 or XENP35612 with CD3 Bispecific AntibodiesIncrease Anti-Tumor Activity In Vivo

In an in vivo study, NSG mice were engrafted intradermally with 2×10⁶pp-65 expressing MDA-MB-231 cells in the right flank on Day −23. On Day−1, mice were engrafted intraperitoneally with 5×10⁶ human PBMCs. Micewere then treated on Days 0, 8, 14, 21, and 28 with a first illustrativeB7H3×CD3 bispecific antibody (CD3bsAb1) (0.5 mg/kg) alone, a secondillustrative B7H3×CD3 bispecific antibody (CD3bsAb2) (0.5 mg/kg) alone,or a combination of XENP34339 (5.0 mg/kg) with CD3bsAb1 or CD3bsAb2.Tumor volumes were monitored by caliper measurements, data for which areshown (days post 1′ dose) in FIGS. 61-62 . Blood was drawn once per weekto investigate lymphocyte expansion, data for which are depicted in FIG.63 for CD45+ cells on Day 14. The data shows that adding CD28costimulation to a CD3 bispecific increases anti-tumor activity in vivo.Notably, CD28 costimulation enables up to a 600-fold increase inlymphocyte expansion.

In another in vivo study, NSG mice that were MHC I/II-DKO (NSG-DKO) andthus resistant to GVHD and another CD3 bispecific (a PSMA×CD3 were used.On Day −7, NSG-DKO mice were inoculated with 5×10⁶ 22RV1 tumor cellseach. On Day 0, mice were engrafted with 5×10⁶ human PBMC cells from arandom donor. Mice were then intraperitoneally treated on Days 0, 7, 14,and 21 with low or high concentration doses of illustrative PSMA×CD3bispecific antibody XENP32220 (sequences as depicted in FIG. 54 ) aloneor in combination with XENP34339. Blood and serum were drawn weekly.Treatment with both the CD3 and the CD28 bsAbs enhanced T cell expansion(as indicated by lymphocyte counts in FIG. 64A-C and specifically Tcells expressing Ki67 proliferation marker in FIG. 64D-E) as well as Tcell activation (as indicated by CD25 and PD1 expression on CD4⁺ andCD8⁺ T cells in FIG. 65 ) in comparison to treatment with the CD3 bsAbalone.

In another study, CD34+ Hu-NSG, which are NSG mice engrafted with humanCD34+ hematopoietic stem cells so as to develop a functional humanimmune system with no reactivity towards the host were obtained from TheJackson Laboratory (Bar Harbor, Me.), were used. On Day −15, mice wereintradermally inoculated with 4×10⁶ pp65-MDA-MB231 cells. Mice were thentreated intraperitoneally on Days 0, 7, and 14 with B7H3×CD3 bsAb alone,XENP35612 alone, B7H3×CD3 bsAb in combination with XENP35612, orB7H3×CD3 bsAb in combination with XENP34339. Tumor volumes weremonitored by caliper measurements, data for which are shown (days post1^(st) dose) in FIGS. 66-67 . Tumor was harvested on Day 23 toinvestigate expansion of tumor infiltrating lymphocytes, data for whichare depicted in FIG. 68 . The data show that XENP35612 combines wellwith CD3 bsAb to suppress tumor growth. Notably, combination ofB7H3×CD28 with B7H3×CD3 enables significantly enhanced anti-tumoractivity in comparison to treatment with B7H3×CD3 alone at earlier timepoints (i.e. days 6 and 9). Additionally, XENP35612 as a single agentsignificantly expands expansion of tumor infiltrating lymphocytes; andcombination of XENP35612 and B7H3×CD3 bsAb significantly enhancesexpansion of tumor infiltrating lymphocytes in comparison to B7H3×CD3bsAb alone.

5F: XENP34339 and XENP37808 are not Superagonistic

Potential superagonistic properties of XENP34339 and XENP37808 wereassessed by air-drying per the Stebbings protocol (Stebbings R. et al.2007). Air-drying of test articles was achieved by drying in a SpeedVac™for 2 hours at room temperature. Human PBMCs were treated for 24 hourswith 10 μg of air-dried XENP34339 or XENP37808, and activity wascompared to the superagonist TGN1412 (XENP29154; sequences for which aredepicted in FIG. 69 ) or PBS control. Airdried TGN1412 promoted IFNγ,IL-6, IL-2, and TNF cytokine secretion from unstimulated human PBMC. Incomparison, the cytokine levels in PBMCs treated with air-driedXENP34339 and XENP37808 remained similar to the negative control of PBS(data shown in FIGS. 70-71 ).

5G: XENP37808 Enhances CD3 Activity of Both Human & Cynomolgus PBMC

In order to investigate whether or not cynomolgus would be a good modelfor toxicology studies, an experiment was performed in which PBMCs from11 unique human donors or 12 unique cynomolgus donors are dosed withXENP37808 in the presence of HEK cells transfected with anti-CD3 scFv(to provide “Signal 1”) at a 10:1 E:T ratio. After 1 or 5 days, IL-2release was measured using MSD assay. The results as depicted in FIG. 72(data from one human donor and one cynomolgus donor) verify thatcynomolgus is a comparably relevant species. Additionally, HEK cellstransfected with anti-CD3 scFv but with B7H3 expression knocked out werealso tested. Consistent with the data in Example 5D, these results showthat XENP37808 enhances CD3 activity of human PBMCs exclusively in thepresence of B7H3.

5H: Additional In Vitro Comparison of XENP34398 and XENP37808

In a first set of experiments, 1,250 22RV1-NLR (having a MESF value of−170K B7H3 antigens) or DU145-NLR cancer cells (having ˜270K B7H3antigens) were seeded per well. After 48 hrs, CD3+ T cells were added atan effector to target ratio of 1:1 with indicated amounts of B7H3×CD3and 1 ug/mL B7H3×CD28, and cell counts were recorded by Incucyte. Theresults, shown in FIG. 73 , demonstrate that in combination with aB7H3×CD3 bispecific, both XENP34398 and XENP37808 induce very similarlevels of RTCC.

In an additional set of experiment 10,000 target cancer cells (OVCAR8having ˜20K B7H3 surface density; 22RV1-NLR having ˜170K B7H3 antigendensity; or DU145-NLR having ˜270K B7H3 antigen density) per well wereseeded. The next day T-cells at an effector to target ratio of 1:1 wereadded with indicated amounts of B7H3×CD28 mAb in the presence of 1 μg/mLof an illustrative B7H3×CD3 bsAb. IL-2 was assayed 24 hours afterseeding. The results shown in FIG. 74 depict the very similar levels offunction as measured by IL-2 induction of XENP34398 and XENP37808 oncell lines of various densities.

Together, these data show that both XENP37808 and XENP34398 display verysimilar activity and are equally efficacious. However, each maypotentially have their own advantages in a clinical setting.

What is claimed:
 1. A heterodimeric antibody that binds to human CD28and human B7H3 comprising: a) a first monomer comprising the amino acidsequence of SEQ ID NO:1019; b) a second monomer comprising the aminoacid sequence of SEQ ID NO:1020; and c) a light chain comprising theamino acid sequence of SEQ ID NO:1021.
 2. A nucleic acid compositioncomprising: a) a first nucleic acid encoding the amino acid sequence ofSEQ ID NO:1019; b) a second nucleic acid encoding the amino acidsequence of SEQ ID NO:1020; and c) a third nucleic acid encoding theamino acid sequence of SEQ ID NO:1021.
 3. An expression vectorcomposition comprising: a) a first expression vector comprising a firstnucleic acid encoding the amino acid sequence of SEQ ID NO:1019; b) asecond expression vector comprising a second nucleic acid encoding theamino acid sequence of SEQ ID NO:1020; and c) a third expression vectorcomprising a third nucleic acid encoding the amino acid sequence of SEQID NO:1021.
 4. A host cell comprising the expression vector compositionof claim
 3. 5. A method of making a heterodimeric antibody that binds tohuman CD28 and human B7H3 comprising culturing the host cell of claim 4under conditions whereby said heterodimeric antibody is expressed andrecovering said heterodimeric antibody.